Grégory Jubelin

Sylvie Pages

Anne Lanois

Marie-Hélène Boyer

Sophie S Gaudriault

Jean-Baptiste Ferdy

Alain Givaudan
email: givaudan@univ-montp2.fr

Sylvie Pagès

Studies of the dynamic expression of the Xenorhabdus FliAZ regulon reveal atypical iron-dependent regulation of the flagellin and haemolysin genes during insect infectione mi_2427 1..14

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Studies of the dynamic expression of the Xenorhabdus

FliAZ regulon reveal atypical iron-dependent regulation of the flagellin and haemolysin genes during insect infection.

Introduction

Xenorhabdus nematophila is a motile Gram-negative bacterium that infects and kills a wide range of insect larvae, including agriculturally relevant species such as the tobacco hornworm, Manduca sexta, and the cotton worm, Spodoptera littoralis [START_REF] Forst | Role of the histidine kinase, EnvZ, in the production of outer membrane proteins in the symbiotic-pathogenic bacterium Xenorhabdus nematophilus[END_REF][START_REF] Sicard | Stages of infection during the tripartite interaction between Xenorhabdus nematophila, its nematode vector, and insect hosts[END_REF]. These enterobacteria also form a speciesspecific mutualistic association with the entomopathogenic nematode Steinernema carpocapsae [START_REF] Thomas | Xenorhabdus gen. nov., a genus of entomopathogenic and nematophilic bacteria of the family Enterobacteriaceae[END_REF][START_REF] Cowles | The Xenorhabdus nematophila nilABC genes confer the ability of Xenorhabdus spp. to colonize Steinernema carpocapsae nematodes[END_REF]. The life cycle of S. carpocapsae nematodes includes reproductive stages occurring exclusively within host insect larvae. The nematode life cycle also includes a developmentally arrested stage termed infective juvenile (IJ). Xenorhabdus nematophila colonizes a discrete intestinal location in the IJ, known as the vesicle [START_REF] Martens | Early colonization events in the mutualistic association between Steinernema carpocapsae nematodes and Xenorhabdus nematophila bacteria[END_REF]. In nature, IJs penetrate insects and release their bacterial symbionts into the host body cavity. The bacteria initially escape insect defence mechanisms by modulating the immune system [START_REF] Ji | An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits the expression of an antibacterial peptide, cecropin, of the beet armyworm, Spodoptera exigua[END_REF][START_REF] Park | Clonal variation in Xenorhabdus nematophila virulence and suppression of Manduca sexta immunity[END_REF]. They then colonize the haemolymph (the insect equivalent of blood) and connective tissues surrounding the anterior midgut, and finally invade the entire body of the larva, leading to its death [START_REF] Sicard | Stages of infection during the tripartite interaction between Xenorhabdus nematophila, its nematode vector, and insect hosts[END_REF]. Bacteria contribute to the mutualistic relationship by establishing and maintaining suitable conditions for nematode reproduction in the insect cadaver [START_REF] Sicard | Effect of native Xenorhabdus on the fitness of their Steinernema hosts: contrasting types of interaction[END_REF].

Xenorhabdus nematophila is also able to kill insects and grow within insect bodies independently of its nematode partner if introduced into the haemolymph by direct injection in the laboratory [START_REF] Poinar | Significance of Achromobacter nematophilus Poinar et Thomas (Achromobacteriaceae, Eubacteriales) in the development of the nematode DD 136[END_REF][START_REF] Sicard | Stages of infection during the tripartite interaction between Xenorhabdus nematophila, its nematode vector, and insect hosts[END_REF].

Xenorhabdus nematophila produces a plethora of putative virulence factors, including toxins, lipases, haemolysins and proteases, which may contribute to pathogenicity and the modification of insect immunity (Forst et al., 1997;[START_REF] Herbert | Friend and foe: the two faces of Xenorhabdus nematophila[END_REF]. In many cases, the inactivation of an individual factor has no effect on overall X. nematophila virulence. Several studies in the last 10 years have revealed the existence of a central regulatory network controlling X. nematophila pathogenesis [START_REF] Goodrich-Blair | Mutualism and pathogenesis in Xenorhabdus and Photorhabdus: two roads to the same destination[END_REF]. The master regulator for flagellar synthesis and motility, FlhDC, has been shown to be required for full virulence in insects and for haemolysin and lipase production [START_REF] Givaudan | flhDC, the flagellar master operon of Xenorhabdus nematophilus: requirement for motility, lipolysis, extracellular hemolysis, and full virulence in insects[END_REF]. FlhDC synthesis is regulated by at least three other transcription factors having therefore pleiotropic effects and affecting virulence in insects [START_REF] Park | Co-regulation of motility, exoenzyme and antibiotic production by the EnvZ-OmpR-FlhDC-FliA pathway in Xenorhabdus nematophila[END_REF][START_REF] Cowles | The global regulator Lrp contributes to mutualism, pathogenesis and phenotypic variation in the bacterium Xenorhabdus nematophila[END_REF][START_REF] Richards | Xenorhabdus nematophila lrhA is necessary for motility, lipase activity, toxin expression, and virulence in Manduca sexta insects[END_REF]. Transcriptional and phenotypic studies of the flagellar cascade clarified the respective roles of motility and other FlhDC-dependent factors in X. nematophila virulence [START_REF] Park | Co-regulation of motility, exoenzyme and antibiotic production by the EnvZ-OmpR-FlhDC-FliA pathway in Xenorhabdus nematophila[END_REF][START_REF] Lanois | FliZ, a flagellar regulator, is at the crossroads between motility, haemolysin expression and virulence in the insect pathogenic bacterium Xenorhabdus[END_REF]. As in Escherichia coli [START_REF] Liu | The FlhD/FlhC complex, a transcriptional activator of the Escherichia coli flagellar class II operons[END_REF], the flagellar class I operon product FlhDC is required for the transcription of class II genes encoding structural components of the flagellar hook-basal body in X. nematophila. FlhDC also controls expression of the alternative sigma factor FliA (s 28 ), which is required for the expression of class III products, including the flagellin monomer FliC [START_REF] Givaudan | flhDC, the flagellar master operon of Xenorhabdus nematophilus: requirement for motility, lipolysis, extracellular hemolysis, and full virulence in insects[END_REF]. In X. nematophila, the FlhD-dependent fliAZ operon has been identified as the key locus mediating the expression of genes involved in lipase, protease and haemolysin activities, motility and full virulence in insects [START_REF] Park | Co-regulation of motility, exoenzyme and antibiotic production by the EnvZ-OmpR-FlhDC-FliA pathway in Xenorhabdus nematophila[END_REF][START_REF] Lanois | FliZ, a flagellar regulator, is at the crossroads between motility, haemolysin expression and virulence in the insect pathogenic bacterium Xenorhabdus[END_REF]. We demonstrated that FliZ controls the haemolysin genes xaxAB and xhlBA and full virulence in insects [START_REF] Lanois | FliZ, a flagellar regulator, is at the crossroads between motility, haemolysin expression and virulence in the insect pathogenic bacterium Xenorhabdus[END_REF]. The two operons directly affected by FliZ encode haemolysins from two families. XaxAB is the prototype of a new extensive family of haemolysins with apoptotic and pore-forming activities in mammalian and invertebrate cells [START_REF] Vigneux | The xaxAB genes encoding a new apoptotic toxin from the insect pathogen Xenorhabdus nematophila are present in plant and human pathogens[END_REF]. The second FliZ-dependent haemolysin, XhlA, is a cell surface-associated haemolysin from a family characterized by a two-partner secretion system [START_REF] Cowles | Expression and activity of a Xenorhabdus nematophila haemolysin required for full virulence towards Manduca sexta insects[END_REF]. Haemolysins XhlA and XaxAB lyse erythrocytes and the two most common types of insect immune cell (granulocytes and plasmatocytes) [START_REF] Brillard | Two distinct hemolytic activities in Xenorhabdus nematophila are active against immunocompetent insect cells[END_REF][START_REF] Cowles | Expression and activity of a Xenorhabdus nematophila haemolysin required for full virulence towards Manduca sexta insects[END_REF].

To increase our understanding of the role of the FliAZ regulon in the life cycle of X. nematophila, investigations of the successive steps of the infectious process and the nematode symbiotic stage are required to determine the timing and sites of regulon expression. In addition, the timely deployment of bacterial determinants requires that Xenorhabdus are able to sense when they have gained entry into the insect haemocoel or into the IJ-stage nematode. Iron is a scarce soluble element in eukaryotic hosts and its low availability often constitute a signal indicating that pathogens have entered inside the host [START_REF] Litwin | Role of iron in regulation of virulence genes[END_REF]. In this study, we showed that expression of the FliA-dependent gene fliC and the FliZdependent genes xaxAB are downregulated under iron starvation conditions in bacterial cultures as well as during the infectious process in living insects. In addition, a high expression level of the FliAZ-dependent genes in insect cadavers is correlated with the increase of iron availability in insect cadavers. These findings highlight the role of iron in regulating the expression of the FliAZ regulon during the insect infection phase of the Xenorhabdus life cycle.

Results

Temporal pattern of expression of the flagellin and FliZ-dependent haemolysin genes in bacterial cultures

Before characterizing the dynamics of the X. nematophila FliAZ regulon in invertebrate hosts, we first investigated the expression patterns of FliA-or FliZ-dependent genes in bacteria cultured in vitro. The FliA-dependent gene fliC, encoding the flagellin monomer, and the FliZ-dependent genes xaxAB and xhlBA, both encoding haemolysins, were used as representative members of the FliAZ regulon. Promoter sequences of these genes were inserted into pPROBE-gfp[AAV], a highly stable vector [START_REF] Miller | Improved gfp and inaZ broad-host-range promoter-probe vectors[END_REF] to create transcriptional fusions with the reporter gene gfp [AAV], which encodes an unstable GFP [START_REF] Andersen | New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria[END_REF]. Constructs were then introduced into the wild-type X. nematophila strain F1 and the patterns of expression of FliAZ-dependent genes were examined during growth in vitro. Fluorescence due to the expression of the P fliC-gfp [AAV] fusion was first detected in the mid-exponential growth phase and peaked early in the stationary phase, subsequently decreasing in the late stationary phase (i.e. after 20 h of growth culture) (Fig. 1A). The temporal pattern of fliC expression is consistent with the bacterial motility observed during the growth of X. nematophila in LB medium (data not shown) [START_REF] Forst | Characterization of the pleiotropic phenotype of an ompR strain of Xenorhabdus nematophila[END_REF]. In contrast expression of the haemolysin genes, xaxAB and xhlBA was not detected before the bacteria reached the stationary phase (Fig. 1A). For confirmation of the pattern of xaxAB expression, we monitored xaxA mRNA synthesis by quantitative RT-PCR (qRT-PCR) and Xax activity for which we have a clear associated phenotype. The number of xaxA transcripts increased rapidly during the early stationary phase as reported for the gfp[AAV] reporter gene (Fig. 1A), and then gradually decreased during late stationary phase (Fig. 1B). The detection of a third of the GFP signal in the medium of a 40-day-old culture of X. nematophila (data not shown) may account for the difference between the two quantification methods, as extracellular GFP is not degraded by specific bacterial proteases. Indeed, after centrifugation, the monitoring of bacterial cell fluorescence displayed a decrease of xax expression from 40 h (data not shown) to a lesser extent than in Fig. 1B. Expression of the xaxAB operon is also correlated, for both induction and extinction, with Xax cytolytic activity, which is detected during the early stationary phase (Fig. 1B) [START_REF] Brillard | Two distinct hemolytic activities in Xenorhabdus nematophila are active against immunocompetent insect cells[END_REF].

Iron availability modulates the expression of flagellin and FliZ-dependent haemolysin genes in vitro

Extensive studies of flagellar gene expression in bacteria have shown that flagellum biosynthesis is affected by many environmental factors (for a review, see [START_REF] Soutourina | Regulation cascade of flagellar expression in Gram-negative bacteria[END_REF]. For the identification of putative stimuli of flagellar and haemolysin gene expression in Xenorhabdus, F1 strains carrying the PfliC-gfp[AAV] or PxaxAB-gfp[AAV] fusion constructs were grown in LB supplemented with various chemicals, and gene expres-sion was monitored by recording GFP fluorescence. Several of the compounds tested had no significant effect on the expression of fliC and xaxAB (Fig. 2). A second class of products [acetylphosphate (ACP) and acetate] enhanced fliC expression without modifying xaxAB transcription. A third class of products significantly modulated expression of the P xaxAB-gfp [AAV] and PfliC-gfp[AAV] constructs (Fig. 2). While glucose had a positive effect on fliC expression and, to a lesser extent on xaxAB expression, p-nitrophenylglycerine (PNPG), a repressor of swarming motility, ethylene- were grown in 96-well plates for 40 h and GFP fluorescence and absorbance at 600 nm were recorded every 30 min. A representative growth curve is indicated by the dashed line. Specific fluorescence is the ratio between GFP fluorescence and absorbance at 600 nm. The results are the means and standard errors of three independent assays. B. Relationship between the amount of xaxA transcript (᭹) and the cytolytic activity of the Xax toxin (D). Xenorhabdus nematophila F1 was grown in LB and xaxA transcript levels were quantified by qRT-PCR. Data are presented as a ratio, with mreB used as the control mRNA. Xax activity is expressed in cytolytic units, as described elsewhere [START_REF] Brillard | Two distinct hemolytic activities in Xenorhabdus nematophila are active against immunocompetent insect cells[END_REF]. The growth curve is indicated by the dashed line.

diamine-tetraacetic acid (EDTA), a divalent cation chelator, and 2,2′-dipyridyl, a chelator more specifically targeting iron, significantly reduced expression of both gfp fusions (Fig. 2).

As iron is essential for the successful establishment of most pathogens in their hosts, we focused on irondependent modulation of the flagellar regulon. We constructed a reporter fusion to monitor iron bioavailability during X. nematophila growth. The gfp[AAV] gene was placed under the control of the promoter sequence from the XNC1-1298 gene, which is the first gene of a locus putatively encoding an iron-chelating siderophore. Based on homology between the protein predicted from XNC1-1298 and the siderophore RhbF [START_REF] Martinez | The organization of intercistronic regions of the aerobactin operon of pColV-K30 may account for the differential expression of the iucABCD iutA genes[END_REF][START_REF] Lynch | Genetic organization of the region encoding regulation, biosynthesis, and transport of rhizobactin 1021, a siderophore produced by Sinorhizobium meliloti[END_REF], the XNC1-1298 gene was renamed xhbF. As expected, expression of the P xhbF-gfp [AAV] reporter construct was clearly detected when 2,2′dipyridyl was added to the culture medium (Fig. 3). In contrast, very weak expression was recorded in LB, in LB supplemented with the non-metal-chelating agent 4,4′-dipyridyl (used as a negative control) or when FeSO4 was added in excess to the medium containing 2,2′-dipyridyl. These data indicate that the PxhbFgfp[AAV] reporter fusion construct is an effective tool for detecting iron depletion. Whereas 4,4′-dipyridyl had no effect on the expression of fliC and xaxAB, 2,2′-dipyridyl significantly decreased the expression of these genes by factors of 3.9 and 2.8 respectively (Fig. 3). The expression levels of fliC and xaxAB were restored by adding excess FeSO4 to LB containing 2,2′-dipyridyl, demonstrating that iron deprivation was responsible for the repression of the fliC and xaxAB genes. The iron dependence of xhlBA regulation was less apparent, as fluorescence levels in the presence of 2,2′-dipyridyl were lower than those in LB alone by a factor of only 1.4 (P-value > 0.01). Iron-dependent modulation of FliAZdependent gene expression was confirmed by qRT-PCR. Indeed, levels of the fliC, xaxA and xhlA transcripts were significantly decreased by factors of 11, 11 and 5, respectively, following the addition of 2,2′dipyridyl to the growth medium (Fig. S1). qRT-PCR experiments also confirmed that the expression of xhbF gene was regulated by iron (Fig. S1). Altogether, these results demonstrate that expression of the FliAZdependent genes in Xenorhabdus requires the presence of iron.

In addition, we investigated the possible direct involvement of Fur, the major regulatory protein modulating iron-dependent gene expression in bacteria [START_REF] Escolar | Opening the iron box: transcriptional metalloregulation by the Fur protein[END_REF] in the iron-dependent expression of FlhDdependent genes in X. nematophila. By carrying out chromatin immunoprecipitation (ChIP) assays with an HA-tagged Fur protein we showed that Fur belongs to the transcriptional complex binding to the promoter region of xhbF. Indeed, the HA-immunoprecipitated DNA fraction was significantly enriched with xhbF promoter region DNA, particularly in presence of iron in growth media (Fig. S2) (compare with the non-target mreB promoter sequence). In contrast, ChIP experiments revealed no specific enrichment in fliC, xaxA, xhlB, fliA and flhD promoter sequences, whatever the growth conditions, showing that Fur does not directly participate to the transcriptional activation of flagellar gene promoters. 1A, except that the LB was supplemented with the various products indicated. Maximal gene expression levels during growth in LB were taken as the standard expression levels (equivalent to 100%). For other growth conditions, results are indicated as a percentage of gene expression in LB. None of the tested conditions affected the growth rate of X. nematophila (data not shown). The results shown are the means and standard errors from three to five independent assays. Significant differences with the standard LB growth condition (P-value < 0.01, Wilcoxon rank sum test) are indicated with asterisks (*).

Qualitative analysis of fliC and xaxAB gene expression during the insect infectious process

In a first attempt, we carried out experiments to compare the behaviour of F1 strains in insect with and without the various gfp[AAV] constructs. As previously shown with other GFP-labelled X. nematophila strains [START_REF] Sicard | Stages of infection during the tripartite interaction between Xenorhabdus nematophila, its nematode vector, and insect hosts[END_REF], tagging X. nematophila with a gfp[AAV] construct, regardless of the precise nature of that construct, had no effect on Xenorhabdus growth kinetics in infected larvae or the LT50 value, which was estimated at 28 h post infection (data not shown). We then investigated the infectious process in detail, by delineating four different stages in the infectious process (Fig. 4A): (i) the early septicaemic stage (ES), when the bacteria grow without killing the insect, (ii) the lethal septicaemic stage (LS), when septicaemia (~10 8 bacteria per insect larva) leads to insect death, (iii) the early cadaveric stage (EC), when about 90% of the larvae are dead, and (iv) the late cadaveric stage (LC), 1 day after insect death.

First, we observed the fluorescence of whole larvae infected with X. nematophila carrying the various gfp[AAV] constructs with macro/zoom optics. While the bioimaging of larvae did not allow detection of fluorescence in whole animal before the LS stage, the fluorescence emitted by late LS stage larvae was evenly distributed throughout the insect body, for all gfp[AAV] constructs (Fig. 4B). We previously showed that the first sites within the insect haemocoel to be colonized by X. nematophila are the haemolymph and the connective tissues surrounding the 1A, except that LB (᭡) was supplemented with 0.15 mM 4,4′-dipyridyl (D), 0.15 mM 2,2′-dipyridyl (᭺) or with 0.15 mM 2,2′-dipyridyl + 0.3 mM FeSO4 (᭹). Specific fluorescence is expressed as the ratio between GFP fluorescence and absorbance at 600 nm. The results are representative of five independent assays. Comparison of gene expression level between growth conditions was realized at the time point of maximal expression in LB + 2,2′-dipyridyl using the statistical model described in Experimental procedures and correcting for differences in microplate reader settings between independent experiments. Significant differences (P-value < 0.01) with the standard LB growth condition were detected for xhbF, fliC, xaxAB in presence of 2,2′-dipyridyl and for xhbF in presence of 2,2′-dipyridyl + FeSO4. No significant difference was detected in presence of 4,4′-dipyridyl.

anterior midgut [START_REF] Sicard | Stages of infection during the tripartite interaction between Xenorhabdus nematophila, its nematode vector, and insect hosts[END_REF]. Epifluorescence microscopy analysis of these two tissues during LS stage revealed that there was no difference in spatial expression pattern between FliAZ-dependent genes and a constitutive reporter, D31 (Fig. 4B). Thus, the expression of flagellar genes is not tissue-specific during insect infection.

We next analysed haemolymph samples of infected larvae at each stage to determine the temporal expression of each gfp construct. We observed that bacteria carrying the PxaxAB-gfp[AAV] construct were not fluorescent at the ES stage (and only few bacterial cells were fluorescent for fliC) whereas bacteria harbouring the D31 or xhbF reporter constructs were (Fig. 4B). Fluorescence from bacteria carrying the PfliC-gfp[AAV] and PxaxAB-gfp[AAV] constructs started to be clearly detected in haemolymph samples during the LS and EC stages, respectively, indicating that flagellin and haemolysin genes were indeed expressed in insect cadavers. Late expression of flagellar genes during the infectious process is in agreement with observation of motile cells and measurement of haemolytic activities in LC stage larvae (data not shown). It is noteworthy that fliC and xaxAB gene expression is heterogeneous at the cellular level in contrast to bacteria carrying the other gfp constructs (Fig. 4B). Altogether, these findings indicate that flagellin and haemolysin gene expression is mostly detected concomitantly to larvae death.

Late expression of the fliC and xaxAB genes is correlated with iron availability in infected insects

Since iron modulates expression level of FliAZ-dependent genes in vitro, we used a quantitative assay of GFP fluorescence to investigate possible interactions between fliC and xaxAB gene expression and iron availability during insect infection. Control experiments were first carried out to determine whether the fluorescence emitted from the constitutive D31 construct was correlated with bacterial counts in the body of the insect in larvae treated or not with an exogenous source of iron. Infected larvae at various stages of infection were individually crushed and fluorescence and bacterial counts were determined for each insect lysate. The fluorescence of the P D31-gfp [AAV] fusion construct was statistically predicted as a linear function of the number of bacteria for all infectious stages (Durbin-Watson test on residuals, DW = 1.672, P-value = 0.1620; see also Fig. 5A). Similar results were obtained in larvae treated with an exogenous source of iron, indicating that iron did not affect expression of PD31gfp[AAV] construct (GLM with gamma-distributed error, F1,98 = 0.655, P = 0.4203 for the overall effect of iron, F2,96 = 0.677, P = 0.5105 for the interaction between stage and iron conditions). Thus, D31 promoter activity remains constant over time in insects and the fluorescence of the PD31-gfp[AAV] fusion construct can be used as an in vivo reference standard.

Thereafter, gene expression was monitored by GFP quantification in infected larvae at each stage of the infectious process after normalization with the fluorescence from the constitutive D31 construct (Fig. 5B). We designed a statistical model in which relative promoter activity at a given infectious stage was compared with that of the previous stage, to determine whether gene expres-sion levels changed over time (see Experimental procedures). To report the iron availability in infected larvae treated or not with iron, we quantified expression of the siderophore enzyme encoding gene xhbF. In larvae without iron supplementation, the activity of the xhbF gene promoter remained high and constant in living or recently deceased animals (stages ES, LS and EC) (Fig. 5B, solid line). Thus, the insect haemolymph fluid, like blood plasma, is an iron-starvation condition for bacterial growth. Furthermore, iron supplementation strongly decreased the level of xhbF gene expression at these stages of infection (Fig. 5B, dashed line), indicating that addition of an exogenous source of iron modulates the iron availability in insects.

Quantification assays using the P fliC-gfp [AAV] and PxaxAB-gfp[AAV] constructs first confirmed the late expression of both genes in infected larvae without iron supplementation (Fig. 5B, solid line). Indeed, xaxAB gene expression was negligible early in the infection but progressively increased between the LS and EC stages of infection (t = 1.759, P-value = 0.0803) and between the EC and LC stages (t = 2.854, P-value = 0.0048), peaking in insect cadavers 2 days after infection. The expression of the fliC gene also gradually upregulated during infection and reached maximal expression in insect cadavers (Fig. 5B). In larvae with iron supplementation, fluorescence emitted by the P xaxAB-gfp[AAV] construct was strongly increased during the ES and LS stages of insect infection (5.1-and 3.6-fold respectively) (Fig. 5B, compare solid and dashed lines). Iron also upregulated the global expression of fliC at the ES stage (twofold, Fig. 5B) without changing the frequency of fluorescent bacteria (30 Ϯ 7% without iron and 28 Ϯ 6% in presence of iron). Thus, artificial increases in the free iron content of infected insects shift the timing of FliAZdependent gene expression, such that the expression of fliC and xaxAB genes is induced before the death of the larvae. During infection without iron supplementation, we also noted that expression of the xhbF gene decreased slightly but significantly (t = 1.708, P-value = 0.0893) between the EC and LC stages (twofold decrease), suggesting that free iron was released after the death of infected insects. Such iron availability may explain the increase of fliC and xaxAB gene expression in dead larvae. Taken together, these findings indicate that the expression of the FliAZ-dependent genes during insect infection is significantly modulated by the availability of iron in infected insects.

FliAZ-dependent genes are not expressed during the symbiotic stage of the X. nematophila life cycle

In natural conditions, X. nematophila is transported into insects by IJ nematodes [START_REF] Martens | Early colonization events in the mutualistic association between Steinernema carpocapsae nematodes and Xenorhabdus nematophila bacteria[END_REF]. To

Real-time expression of FliAZ-dependent genes 7 © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology determine whether activation of the FliAZ regulon was restricted to the insect cadaver stage, S. littoralis larvae were infested with S. carpocapsae IJs associated with F1 strains carrying the various gfp[AAV] constructs (see Experimental procedures for bacterium-nematode recombination). IJs were collected after their emergence from insect cadavers 9 days after infestation. Epifluorescence microscopy studies revealed that more than 70% of the IJs recently emerging from insects infected with the F1 (PD31-gfp[AAV]) strain had fluorescent bacteria in their vesicles (Fig. 6). In contrast, no fluorescence was detected from bacteria within IJs emerging after association with F1 strains carrying PfliC-gfp[AAV], PxaxABgfp[AAV], PxhlBA-gfp[AAV] or PxhbF-gfp[AAV] constructs (Fig. 6). We then monitored gfp[AAV] fusion construct expression over time in S. littoralis larvae infested with IJs associated with GFP-tagged bacteria. As in the injection assays, flagellin and haemolysin gene expression was first detected by epifluorescence microscopy at the time of insect death, whereas expression of the constitutive reporter PD31-gfp[AAV] or PxhbF-gfp[AAV] was recorded as early as the ES stage (data not shown). Thus, the expression of fliC, xhlBA and xaxAB occurred specifically at the time of death of the insect, regardless of the route of infection (direct injection or nematode infestation). These findings demonstrate that the flagellar regulon is not expressed in the nematode vesicle, suggesting that iron is not limited in this specialized compartment. Fig. 5. Expression of the flagellin and haemolysin genes is temporally regulated during insect infection, as a function of iron availability. For each experiment, at time zero, we injected 10 4 bacteria into the haemocoel of the insect larvae. A. Relationship between the fluorescence obtained with the PD31-gfp[AAV] construct and the number of bacteria in insects. The dashed curve corresponds to a generalized linear model, with a gamma-distributed error and an identity link function. B. FliAZ-dependent gene expression is modulated by free iron availability in infected insects. Each figure represents the log-transformed ratio of the fluorescence activity of the xaxAB, fliC or xhbF constructs to that of the control D31 construct in larvae with and without iron supplementation (dashed lines and solid lines respectively). A value of zero thus indicates that the construct had a similar level of expression to the control D31 construct (dotted lines). Vertical bars are two times the standard error of the estimate. The triangles pointing upward indicate that the expression of the construct has increased since the previous stage; downward-pointing triangles indicate that the expression has decreased since the previous stage. The level of significance is indicated by whether the symbol is grey or white (grey: P-value is below 0.01; white, P-value is below 0.1). Open circles indicate no change in the expression of the gene since the previous stage (i.e. P-value > 0.1). The first stage, for which this test cannot be performed, is represented by open squares. The P-values indicated at the top of each graph correspond to a comparison between the relative activities obtained in insect larvae with and without iron supplementation. The numbers below the P-values are the sample size in this test, the first number indicating the number of measurements without iron supplementation, and the second number indicating the number of measurements with iron supplementation.

Discussion

FliA-dependent flagellin expression and FliZ-dependent haemolysin expression are restricted to the insect cadaver phase

The X. nematophila life cycle has been divided into three phases: insect infection, bacterial and nematode reproduction in the insect cadaver, and nematode transmission of X. nematophila to insect hosts [START_REF] Richards | Masters of conquest and pillage: Xenorhabdus nematophila global regulators control transitions from virulence to nutrient acquisition[END_REF]. Gene inactivation studies have identified regulatory proteins that alter virulence in insects and affect expression of the flagellar regulon [START_REF] Givaudan | flhDC, the flagellar master operon of Xenorhabdus nematophilus: requirement for motility, lipolysis, extracellular hemolysis, and full virulence in insects[END_REF][START_REF] Goodrich-Blair | Mutualism and pathogenesis in Xenorhabdus and Photorhabdus: two roads to the same destination[END_REF][START_REF] Richards | Xenorhabdus nematophila lrhA is necessary for motility, lipase activity, toxin expression, and virulence in Manduca sexta insects[END_REF][START_REF] Herbert | CpxRA influences Xenorhabdus nematophila colonization initiation and outgrowth in Steinernema carpocapsae nematodes through regulation of the nil locus[END_REF]. Among flagellar genes, the fliAZ operon has been identified as the key locus mediating the expression of genes involved in motility, lipase, protease and haemolysin activities [START_REF] Park | Co-regulation of motility, exoenzyme and antibiotic production by the EnvZ-OmpR-FlhDC-FliA pathway in Xenorhabdus nematophila[END_REF] and full virulence in insects [START_REF] Lanois | FliZ, a flagellar regulator, is at the crossroads between motility, haemolysin expression and virulence in the insect pathogenic bacterium Xenorhabdus[END_REF]. Xenorhabdus must be able to control the expression of this gene network, to enable it to cope with the shifts in host environment inherent to the successful colonization of two invertebrate hosts. In this study, we observed that, regardless of the route of infection (bacterial injection or infestation by nematobacterial complexes), expression of the FliA-dependent flagellin gene fliC and the FliZ-dependent haemolysin genes xaxAB and xhlBA was high in dead insects whereas it remained negligible in living insects until the advanced stages of septicaemia. We also found that these fliAZ-dependent genes were not expressed in IJ nematodes. Activation of the flagellar cascade in dead insects may allow bacteria to move through cadaver towards nutrient-rich niches by chemotaxis, facilitating their rapid growth. Other activities encoded by the flagellar regulon (e.g. lipases, proteases and haemolysins) are also thought to contribute to the degradation of insect tissues, releasing nutrients for use by the nematodes. Consistent with this hypothesis, nematodes colonized with the X. nematophila xlpA mutant display delayed IJ emergence and produce significantly fewer IJ progeny in total than nematodes colonized with wild-type strain, suggesting that the FliA-dependent lipase XlpA is involved in degrading insect lipids, thereby supporting nematode production [START_REF] Richards | Examination of Xenorhabdus nematophila lipases in pathogenic and mutualistic host interactions reveals a role for xlpA in nematode progeny production[END_REF]. As X. nematophila must assemble the flagellar secretion system before it can secrete XlpA lipase [START_REF] Park | Co-regulation of motility, exoenzyme and antibiotic production by the EnvZ-OmpR-FlhDC-FliA pathway in Xenorhabdus nematophila[END_REF] and initiate motility, hydrolytic enzyme secretion may coincide with the dispersal of bacteria within insect cadavers.

Iron availability controls flagellar cascade expression in X. nematophila

An in vitro screening of conditions modulating the expression of FliAZ-dependent genes revealed that iron limitation drastically inhibits expression of both fliC and xaxAB genes. Iron signal may act on the flagellar cascade through the class I flhDC operon because expression of the flhDC operon and the FlhDC-dependent class II flagellar gene flgB were repressed in presence of the ironchelator 2,2′-dipyridyl, by factors of 2 and 10 respectively (data not shown). The flagellar cascade and iron regulation have both been studied in detail in bacteria, but iron has rarely been reported to modulate bacterial motility [START_REF] Mccarter | Iron regulation of swarmer cell differentiation of Vibrio parahaemolyticus[END_REF][START_REF] Guzzo | Transcription of the Escherichia coli fliC gene is regulated by metal ions[END_REF][START_REF] Deziel | rhlA is required for the production of a novel biosurfactant promoting swarming motility in Pseudomonas aeruginosa: 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs), the precursors of rhamnolipids[END_REF]. By contrast to few data for motility, the stimulatory effect of iron starvation conditions on the production of haemolysins and toxins has been widely described in bacterial pathogens (for a review, see [START_REF] Litwin | Role of iron in regulation of virulence genes[END_REF]. Conversely, we show here an original iron regulation of haemolysin genes since both FliZ-dependent haemolysin genes, xaxAB and xhlBA, are repressed in iron-limiting conditions in X. nematophila. A previous study reported that the xhlBA operon in X. nematophila was expressed three times more strongly in iron starvation conditions [START_REF] Cowles | Expression and activity of a Xenorhabdus nematophila haemolysin required for full virulence towards Manduca sexta insects[END_REF]. The discrepancy between the results of these two studies may be accounted for (i) by the use in Cowles' study of a different X. nematophila strain and (ii) by the determination of xhlA mRNA levels in exponentially growing cells, in which xhlBA expression levels are very low, being no more than 1/10 that in the stationary phase of growth. To our knowledge, Xax and XhlA from X. nematophila are the first haemolysins to be described for which expression is inhibited by iron limitation. Although contrasting with the typical iron starvation-induced haemolysins involved in bacterial pathogen virulence, the downregulation of xaxAB and xhlBA gene expression in X. nematophila in the absence of iron is not surprising if we consider that these two haemolysins may have a role during the insect cadaver phase as recently demonstrated for another FliAZ-dependent genes [START_REF] Richards | Examination of Xenorhabdus nematophila lipases in pathogenic and mutualistic host interactions reveals a role for xlpA in nematode progeny production[END_REF].

Iron may constitute a signal for the transition between the insect infection and nematode reproduction phases

Organisms, including insects, have evolved mechanisms for stabilizing the iron availability through the control of its uptake, transport and storage [START_REF] Locke | Iron economy in insects: transport, metabolism, and storage[END_REF]. Insect pathogenic bacteria have therefore developed a number of diverse mechanisms for scavenging iron from the host. Indeed, a recent study showed that the mutation of exbD in the entomopathogenic bacteria Photorhabdus, a gene encoding a component of the TonB system involved in iron uptake, leads to slower growth in insects, demonstrating a requirement for iron uptake for full virulence [START_REF] Watson | The exbD gene of Photorhabdus temperata is required for full virulence in insects and symbiosis with the nematode Heterorhabditis[END_REF]. Similarly, we found that expression of the siderophore-related gene xhbF was strongly upregulated upon the infection of larvae by X. nematophila, indicating that iron uptake systems are triggered during Xenorhabdus-insect interactions. In addition to fulfilling metabolic requirements, iron also regulates the expression of the FliAZ regulon during insect infection. Indeed, artificial increases in the free iron content of infected insects shift the timing of fliC and xaxAB expression, such that these genes are highly expressed in living larvae. Combined with inhibition of xhbF expression in iron-treated larvae, these results indicate that iron sensing by Xenorhabdus is required to establish the sequential expression of siderophore and flagellar genes during insect infection. In the absence of iron supplementation, the increase of xaxAB gene expression was partially correlated to the slight increase in iron availability. Most of the iron in insects is either linked to serum transferrins and ferritins circulating in the haemolymph or stored in cells, including enterocytes in the midgut in particular [START_REF] Nichol | Iron metabolism in insects[END_REF]. Interestingly, iron-rich sites are the first areas within the insect haemo-coel to be colonized by X. nematophila [START_REF] Sicard | Stages of infection during the tripartite interaction between Xenorhabdus nematophila, its nematode vector, and insect hosts[END_REF]. The expression dynamics of FliAZ-dependent genes may be related to sequential iron release during the final stages of insect infection. Large amounts of bacterial enzymes, such as proteases, may be produced just before the death of the insect, and their action begins to release free iron, through the proteolysis of extracellular iron-transport proteins. A second wave of iron release may then take place after cell lysis and midgut degradation, leading to the maximal levels of FliAZ-dependent gene expression observed during the insect cadaver stage. Iron may also result from the lysis of a part of the bacterial population in insects.

In conclusion, the flagellin gene fliC, and haemolysin genes xaxAB and xhlBA, controlled by the FliA and FliZ regulators, respectively, are specifically expressed at the time of the death of the infected insect and may be required for bacterial dissemination and tissue degradation in the cadaver, facilitating efficient nematode reproduction. Iron availability may constitute a signal for the upregulation of flagellin and haemolysin gene expression during the transition between the insect infection and nematode reproduction stages. This signalling pathway is part of a complex regulatory network tightly controlling the expression of specific sets of genes required for transitions between the symbiotic and pathogenic lifestyles of X. nematophila.

Experimental procedures

Bacterial strains, plasmids and growth conditions

The strains and plasmids used in this study are listed in Table S1. Bacteria were grown routinely in Luria-Bertani (LB) medium or Mot broth (1% tryptone, 0.5% NaCl, 10 mM MgSO4) at 28°C (X. nematophila) or 37°C (E. coli). Kanamycin (20 mg l -1 ), chloramphenicol (15 mg l -1 ), tetracycline (7.5 mg l -1 ), 2,2′-dipyridyl (0.15 mM), 4,4′-dipyridyl (0.15 mM) and FeSO4 (0.3 mM) were added to the medium as required.

Molecular genetic techniques and RNA preparation

DNA manipulations were carried out as previously described [START_REF] Ausubel | Current Protocols in Molecular Biology[END_REF]. Plasmids were introduced into E. coli by transformation and transferred to X. nematophila by conjugative mating [START_REF] Givaudan | flhDC, the flagellar master operon of Xenorhabdus nematophilus: requirement for motility, lipolysis, extracellular hemolysis, and full virulence in insects[END_REF]. All constructs were sequenced by Millegen (Labège, France). The primers used in this study (Eurogentec) are described in Table S2.

Total RNA was extracted with TRIzol reagent, according to the manufacturer's instructions (Invitrogen), and purified with the High Pure RNA Isolation kit (Roche), including incubation with DNase I. RNA concentration was determined by measuring absorbance at 260 nm. For every RNA preparation, we assessed DNA contamination by carrying out a control PCR before qRT-PCR analysis. We constructed a plasmid for the constitutive expression of gfp, using the D3 promoter region previously identified in the screening of a X. nematophila promoter library, which we inserted into pBBR1-KGFP upstream from gfp mut3 [START_REF] Sicard | Stages of infection during the tripartite interaction between Xenorhabdus nematophila, its nematode vector, and insect hosts[END_REF]. For this study, we inserted the 1.1 kb HindIII blunt end-XbaI fragment containing the D3 promoter region from the D3 plasmid between the SalI blunt end and XbaI sites of pPROBE-gfp[AAV] to yield PD31-gfp [AAV].

Analysis of the expression of the gfp[AAV] constructs in vitro

We studied the kinetics of gene expression in vitro, by inoculating 180 ml of LB supplemented with kanamycin in blacksided, clear-bottomed 96-well plates (Greiner) with 20 ml of a 1/50 dilution of an overnight culture of the F1 strain carrying the various GFP constructs. The high total dilution factor (1/500) ensured that GFP proteins from the pre-culture were diluted to below the threshold of detection before cell divisions and did not interfere with the monitoring of gene expression. Plates were then incubated for 40 h at 28°C, with orbital shaking, in an Infinite M200 microplate reader (Tecan). Absorbance at 600 nm and GFP fluorescence intensity, with excitation at 485 Ϯ 4.5 nm and emission at 520 Ϯ 10 nm, were measured every 30 min. Specific fluorescence was obtained by dividing fluorescence units by the absorbance value. We analysed the effects of various chemical compounds on xaxAB and fliC expression. Experiments were carried out as described above, except that LB was supplemented with 4,4′-dipyridyl, FeSO4, or the products listed in Fig. 2.

Insect infection assays and GFP fluorescence recordings

The common cutworm, S. littoralis, was reared on an artificial diet, at 23°C, with a photoperiod of 12 h. Fifth-instar larvae were selected.

Infection of insect larvae with the X. nematophila gfp[AAV] constructs. Experiments were performed as previously described [START_REF] Sicard | Stages of infection during the tripartite interaction between Xenorhabdus nematophila, its nematode vector, and insect hosts[END_REF]. Briefly, the surface of the insect larva was sterilized with 70% (v/v) ethanol before the injection of bacteria into the haemocoel. We then used a Hamilton syringe to inject groups of 20 larvae with 20 ml each of the appropriate dilution of bacterial cultures grown for 20 h. Six independent experiments were performed for each strain. Treated larvae were incubated individually for up to 48 h and the time at which the insect died was recorded. A mean of 99.3% of bacteria retained the pPROBE plasmids 48 h after injection into insect larvae. We evaluated bacterial growth during insect infection, by counting bacteria within the insect body at various time points after injection. We removed the cuticles from five insect larvae per time point and crushed the soft larval bodies in a homogenizer (Ultra-Turrax™). A low-speed centrifugation (2000 g) was performed to remove large pieces of tissue and the supernatants were collected. Samples were serially diluted and bacteria were counted under the microscope, using a Thoma-type counting chamber, at each stage of the infection process.

Quantification of bacterial gene expression in S. littoralis. We evaluated promoter activity in the infective bacterial population, by recording the fluorescence of soft larval body extracts (described above) on an Infinite M200 microplate reader (Tecan). We analysed 5-20 larvae from at least two independent experiments per fusion and per infection stage. Fluorescence values were subtracted from the autofluorescence values obtained for insects (at the same infection stage) infected with bacteria harbouring the promoterless pPROBEgfp [AAV] vector. The resulting values were normalized with respect to the fluorescence values obtained for the PD31gfp[AAV] constitutive reporter fusion construct used as a reference (see Statistical analysis). For experiments involving iron supplementation, we simultaneously injected bacteria and 0.4 mg of FeSO4 into the insect haemolymph. The monitoring of larval survival after iron supplementation revealed no effect on insect health.

Real-time imaging of larval fluorescence during infection. We monitored the spatial patterns of gene expression in whole infected insect larvae, with a fluorescence macroscope (Olympus™ MVX10 MacroView). Fluorescence imaging was performed with a 6 megapixel digital camera (CP6-CS, M42optic), including ultra-fast, full-resolution live image preview transformation software (MicrosCap-3, Spectrum software). Insect larvae were also dissected and fluorescent bacteria were localized as previously described [START_REF] Sicard | Effect of native Xenorhabdus on the fitness of their Steinernema hosts: contrasting types of interaction[END_REF].

Statistical analysis

We first analysed expression of the control fusion PD31gfp[AAV] construct during the four stages of infection. If promoter activity is constant during the infectious process, as expected, then fluorescence values should increase over time, in proportion to the number of bacterial cells counted in the insect larvae. We therefore used R statistical software to generate a generalized linear model (GLM) predicting the fluorescence of the control construct as a linear function of the estimated number of bacterial cells in the larvae. We assumed that the fluorescence data followed a gamma distribution and we used the identity link function. We then tested the hypothesis that fluorescence variation resulted solely from variations in the number of bacterial cells, by inspecting the residuals of this model. In particular, we checked for potential autocorrelation in the residuals that might result from non-linearity, using a Durbin-Watson test for autocorrelation.

For the detection of up-or downregulation of expression of the xhbF, fliC and xaxAB constructs with respect to the control D31 construct, we generated a GLM fitting all fluorescence data, again assuming that fluorescence followed a gamma distribution, this time using a log link function. We first adjusted the GLM to predict the average log-transformed fluorescence of a gene x, log ε, e being a gamma-distributed error term. The adjusted variable δ t x +1 thus quantifies the difference in expression between a gene and the control construct. We used the same sort of statistical modelling to check for effects of iron supplementation on the level of expression of each construct. For the detection of up-or downregulation between infection stages for a given construct, we fitted a GLM with the same overall structure as that described above, but in which fluorescence value of a gene at a particular stage was predicted from that of the control D31 construct and from the fluorescence value of the gene at the previous stage: log

log F F F F t x t t t x t x + + + ( ) = ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ + + 1 1 1 c c λ ε.
In this model, we first predicted the fluorescence for a given gene if the expression of that gene had not changed since the previous stage. This prediction was obtained by multiplying the fluorescence of the control construct at stage t + 1 by the ratio of the fluorescence of the gene to that of the control construct at stage t. We then added a quantity λ t x +1 to this first prediction. The adjusted variable λ t x +1 thus quantifies the change in expression of gene x between stages t and t + 1.

Insect infestation with S. carpocapsae containing gfp[AAV]-expressing X. nematophila Axenic nematodes of S. carpocapsae (SK27, France) were reared, as previously described [START_REF] Sicard | Stages of infection during the tripartite interaction between Xenorhabdus nematophila, its nematode vector, and insect hosts[END_REF], from an in vitro S. carpocapsae production system, after egg sterilization. Living axenic offspring (IJs) were maintained in the laboratory by passage through larval-stage Galleria mellonella (wax moth) and harvested on White traps [START_REF] White | A method for obtaining infective nematode larvae from cultures[END_REF]. We produced GFP-labelled bacterium-nematode complexes, using an in vivo technique [START_REF] Sicard | Effect of native Xenorhabdus on the fitness of their Steinernema hosts: contrasting types of interaction[END_REF] in which the insect larvae were infected with bacterial constructs and axenic IJ nematodes. About 700 axenic IJs were first placed on a piece of filter paper in a Petri dish and incubated with 10 larvae of S. littoralis at room temperature. After 24 h of infestation, insect larvae were injected with 20 ml (~2000 bacteria) of bacteria harbouring gfp[AAV] constructs that had been grown for 20 h. After the insects had died (48 h post infestation), cadavers were transferred to White traps. Fourteen days after infestation, the IJs emerging from the cadavers were collected in Ringer's solution. These GFPlabelled bacterium-nematode complexes were then used to infest new S. littoralis larvae. Emergence (i.e. IJ production) was observed 9 days after infestation, for each bacterial construct. We used a Leica DMR microscope to visualize fluorescent bacteria in the vesicles of freshly emerged IJs. We checked the stability of bacterial plasmids after the second generation of IJs, by streaking appropriate dilutions of IJ extracts onto selective plates [START_REF] Sicard | Stages of infection during the tripartite interaction between Xenorhabdus nematophila, its nematode vector, and insect hosts[END_REF]. For each GFP-labelled strain, more than 93% of the bacteria recovered from crushed IJs were found to be kanamycinresistant, indicating that the GFP-encoding plasmids persisted in the bacterial symbionts 9 days after infestation.

qRT-PCR analysis

Quantitative RT-PCR was performed in two steps. First, cDNAs were synthesized from 1 mg of total RNA from Xenorhabdus F1 cultured in Mot broth alone or Mot broth supplemented with 2,2′-dipyridyl or with 2,2′dipyridyl + FeSO4, using the Super Script II Reverse Transcriptase from Invitrogen and random hexamers (100 ng ml -1 ) from Roche Diagnostics. qPCR was performed in triplicate with the LightCycler 480 SYBR Green I Master kit from ROCHE Diagnostics, with 1 ml of cDNA synthesis mixture and 1 mM of specific gene primers for xaxA, xhlA, fliC, xhbF, sodB and exbB. The enzyme was activated by heating for 10 min at 95°C. Reactions were performed in triplicate, using 45 cycles of 95°C for 5 s, 60°C for 5 s and 72°C for 10 s, and were monitored with the Light Cycler 480 system (Roche). Melting curves were analysed for each reaction and each curve contained a single peak. Amounts of PCR products were calculated from standard curves obtained for PCR with serially diluted genomic DNA from X. nematophila F1. All data are presented as a ratio, with mreB used as the control gene (95% confidence limits).

ChIP assay

The Fur-HA protein was generated by using the KpnLfur-HA (harbouring the ribosome binding site region of fliC and the first 12 codons of the fur sequence) and BamRfur-HA (designed to insert the HA sequence immediately upstream from the stop codon of the fur gene) primers to amplify a fur-HA PCR fragment from F1 genomic DNA. This PCR fragment was then inserted into pBBR1-MCS2 digested with KpnI and BamHI. The resulting plasmid, pBB-Fur-HA, was transferred into the X. nematophila F1 strain and ChIP assays were performed, as previously described [START_REF] Lanois | FliZ, a flagellar regulator, is at the crossroads between motility, haemolysin expression and virulence in the insect pathogenic bacterium Xenorhabdus[END_REF]. Briefly, the X. nematophila strain producing Fur-HA was cultured (to an OD540 of 1.2) in Mot broth or Mot broth supplemented with 2,2′-dipyridyl or with 2,2′dipyridyl + FeSO4. We then incubated 10 ml of cultures in the presence of 1% formaldehyde for 30 min at room temperature and stopped the reaction by adding 125 mM glycine and incubating for a further 5 min. Cells were collected by centrifugation, washed twice with cold phosphate-buffered saline and lysed, and the resulting cell extracts were sonicated to obtain DNA fragments with a mean size of 600 bp. For the immunoprecipitation of Fur-cross-linked DNA, we incubated 700 ml of the extract with 7 ml of rabbit anti-HA Tag (Sigma) at room temperature for 90 min. Samples were incubated with Protein G Sepharose 4 fast flow (Amersham) for 1 h at room temperature and then centrifuged; 50 ml of supernatant was removed for the preparation of total DNA (input DNA). Beads were then washed in several buffers and the immunoprecipitated material was eluted with 100 ml of elution buffer. After proteinase K treatment, cross-linking in immunoprecipitated and total DNA was reversed by incubation at 67°C for 8 h. Immunoprecipitated and total DNA were then purified with the QIAquick PCR Purification kit (Qiagen). The enrichment of the immunoprecipitated material with specific DNA fragments was determined by qPCR on a LightCycler 480 system (Roche), using the LightCycler 480 SYBR Green I Master kit and promoter-specific primers for the xhbF, xaxA, xhlB, fliC, flhD and fliA genes and internal primers for the mreB and 16S genes.

Fig. 1 .

 1 Fig. 1. Expression of the fliC, xaxAB and xhlBA genes during the growth in vitro of X. nematophila. A. F1 strains carrying the PfliC-gfp[AAV], PxaxAB-gfp[AAV] or PxhlBA-gfp[AAV] construct or the promoterless vector pPROBE-gfp[AAV] were grown in 96-well plates for 40 h and GFP fluorescence and absorbance at 600 nm were recorded every 30 min. A representative growth curve is indicated by the dashed line. Specific fluorescence is the ratio between GFP fluorescence and absorbance at 600 nm. The results are the means and standard errors of three independent assays. B. Relationship between the amount of xaxA transcript (᭹) and the cytolytic activity of the Xax toxin (D).Xenorhabdus nematophila F1 was grown in LB and xaxA transcript levels were quantified by qRT-PCR. Data are presented as a ratio, with mreB used as the control mRNA. Xax activity is expressed in cytolytic units, as described elsewhere[START_REF] Brillard | Two distinct hemolytic activities in Xenorhabdus nematophila are active against immunocompetent insect cells[END_REF]. The growth curve is indicated by the dashed line.

Fig. 2 .

 2 Fig. 2. Effects of various chemical compounds on the expression of fliC and xaxAB in vitro. F1 strains carrying the PfliC-gfp[AAV] or PxaxAB-gfp[AAV] construct were grown as shown in Fig.1A, except that the LB was supplemented with the various products indicated. Maximal gene expression levels during growth in LB were taken as the standard expression levels (equivalent to 100%). For other growth conditions, results are indicated as a percentage of gene expression in LB. None of the tested conditions affected the growth rate of X. nematophila (data not shown). The results shown are the means and standard errors from three to five independent assays. Significant differences with the standard LB growth condition (P-value < 0.01, Wilcoxon rank sum test) are indicated with asterisks (*).

Fig. 3 .

 3 Fig. 3. Iron depletion inhibits the expression of FliAZ-dependent genes. F1 strains carrying the PfliC-gfp[AAV], PxaxAB-gfp[AAV], PxhlBA-gfp[AAV] or positive control PxhbF-gfp[AAV] construct were grown as shown in Fig.1A, except that LB (᭡) was supplemented with 0.15 mM 4,4′-dipyridyl (D), 0.15 mM 2,2′-dipyridyl (᭺) or with 0.15 mM 2,2′-dipyridyl + 0.3 mM FeSO4 (᭹). Specific fluorescence is expressed as the ratio between GFP fluorescence and absorbance at 600 nm. The results are representative of five independent assays. Comparison of gene expression level between growth conditions was realized at the time point of maximal expression in LB + 2,2′-dipyridyl using the statistical model described in Experimental procedures and correcting for differences in microplate reader settings between independent experiments. Significant differences (P-value < 0.01) with the standard LB growth condition were detected for xhbF, fliC, xaxAB in presence of 2,2′-dipyridyl and for xhbF in presence of 2,2′-dipyridyl + FeSO4. No significant difference was detected in presence of 4,4′-dipyridyl.

Fig. 4 .

 4 Fig. 4. Temporal and spatial expression of the flagellin and haemolysin genes during different stages of insect infection. For each experiment, at time zero, we injected 10 4 bacteria into the haemocoel of the insect larvae. A. Log-transformed numbers of bacterial cells are indicated for each of the four infection stages (see the text for explanation of the infection stages). For each stage, the horizontal thick line indicates the median number of cells, and the box corresponds to the range within which 75% of the observations fall. The error bars correspond to the range of observed values once potential outliers (open squares) are excluded. Potential outliers are defined as values that deviating by more than 1.5 times the box height from the box limit. Diamonds indicate the proportion of surviving insects for each of the four stages. hpi, hours post infection. B. In the upper panel, whole larvae from late LS stage were observed with a fluorescence macroscope, with an exposure time set to 600 ms for photography (bars, 2.5 mm). The LS stage larvae were also dissected and connective tissues were observed by fluorescence microscopy (bars, 5 mm). Other panels below depict haemolymph samples from ES, LS and EC stage larvae infected with F1 strains carrying the indicated gfp constructs observed by fluorescence microscopy. Pictures were taken using both phase-contrast and GFP fluorescence illumination (bars, 5 mm).

Fig. 6 .

 6 Fig. 6. The flagellin and haemolysin genes are not expressed in X. nematophila bacteria associated with IJ nematodes. Axenic nematodes of S. carpocapsae were associated with F1 strains carrying PD31-gfp[AAV], PfliC-gfp[AAV], PxhlBA-gfp[AAV], PxaxAB-gfp[AAV] or PxhbF-gfp[AAV] constructs. Larvae of S. littoralis were infested with the various bacterium-nematode complexes and IJs were observed by fluorescence microscopy shortly after their emergence from insect cadavers. Nematode-carrying bacteria harbouring the PD31-gfp[AAV] construct were also photographed in phase contrast (upper left picture). Arrows indicate the bacterium-containing vesicle of the IJs. Bars represent 30 mm.

  Construction of plasmids expressing gfp[AAV] under the control of the fliC, xaxAB, xhlBA or xhbF gene promoters or a constitutive promoter (D31) We obtained plasmids expressing the reporter gene gfp[AAV] under the control of gene promoters, by amplifying DNA fragments (~200-450 bp) located upstream from fliC, xaxA, xhlB and xhbF by PCR from F1 genomic DNA, with primers containing a KpnI or BamHI restriction site. The PCR products were digested and inserted into the corresponding sites of pPROBE-gfp[AAV] or pPROBE′-gfp[AAV] vectors, yielding PfliC-gfp[AAV], PxaxAB-gfp[AAV], PxhlBA-gfp[AAV] and PxhbFgfp[AAV].

G. Jubelin et al. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology

© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology

G. Jubelin et al. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology

G. Jubelin et al. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology

G. Jubelin et al. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology

Acknowledgements

We thank Nadège Ginibre and Hélène Girard for technical assistance. We also thank the Montpellier RIO Imaging facility for the use of their fluorescence macroscope and Philippe Clair from Montpellier GenomiX facility for expert technical assistance with real-time PCR. G.J. was supported by a 'jeune docteur' grant from INRA and this work was supported by Grant No. 2009_1133_01 from INRA.

Supporting information

Additional Supporting Information may be found in the online version of this article: Total RNA from stationary-phase cultures (OD600 of 1.4) was extracted and used for qRT-PCR analysis with specific internal primers for the xhbF, xaxA, xhlA and fliC genes. Quantitative PCR was performed in triplicate and data are presented as a ratio, with mreB used as the control gene. Values are means of three independent assays and were compared using Student's t-test (95% confidence limits). Significant differences with the standard rich media condition (P-value < 0.05) are indicated with asterisks (*). 2). Relative enrichment with the indicated promoter is expressed after normalization with respect to a control region in the 16S gene. Values are means Ϯ the standard errors of three independent assays and were compared using unilateral Wilcoxon test. For each growth condition, significant enrichments (P-value < 0.01) of gene promoter region relative to mreB are indicated with asterisks (*). Table S1. Bacterial strain and plasmids used in this study. Table S2. Oligonucleotides used in this study.

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.


C Desbiez
email: desbiez@avignon.inra.fr

C Chandeysson

H Lecoq

B Moury

A simple, rapid and efficient way to obtain infectious clones of potyviruses

Keywords: plant ssRNA virus, potyvirus, infectious clone, intron, cloning in yeast

The availability of an infectious cDNA clone is a prerequisite for genetic studies on RNA viruses. However, despite important improvement in molecular biology techniques during the last decades, obtaining such clones often remains tedious, time-consuming and rather unpredictable. In the case of potyviruses, cDNA clones are frequently unstable due to the toxicity of some viral proteins for bacteria. The problem can be overcome by inserting introns into the viral sequence but this requires additional steps in the cloning process and depends on the availability of suitable restriction sites in the viral sequence or adjunction of such sites by mutagenesis. Homologous recombination in yeast rather than in vitro restriction and ligation can be used to build infectious clones or other viral constructs. This paper describes how, by using recombination in yeast and fusion PCR, infectious intron-containing clones were obtained within a few weeks for two strains of watermelon mosaic virus (WMV, Potyvirus), whereas previous attempts using "classical" cloning techniques had failed repeatedly. Using the same approach, intronless infectious clones of two other potyviruses, zucchini yellow mosaic virus (ZYMV) and papaya ringspot virus (PRSV), were obtained in less than two weeks.

Comment citer ce document : Desbiez, C. (Auteur de correspondance), Chandeysson, C., Lecoq, H., [START_REF] Desbiez | A simple, rapid and efficient way to obtain infectious clones of potyviruses[END_REF] The molecular tools for manipulating RNA remain scarce. Infectious cDNA clones have therefore been developed in the last 30 years for genetic studies of plant RNA viruses. For potyviruses, that represent about 25% of all known plant viruses, the first infectious clone was obtained in 1989 [START_REF] Domier | Infectious in vitro transcripts from cloned cDNA of a potyvirus, tobacco vein mottling virus[END_REF]. More than 20 years later, about 40 clones belonging to 15 different virus species are now available, and such tools are still missing for many agronomically important viruses. Among the difficulties relevant to the building of potyvirus infectious clones are the length of the viral RNA (about 10 kilobases, making one-step cloning poorly efficient), the highly deleterious effects of adding any extra nucleotides in the 5' or 3' extremity of the genome during the cloning process [START_REF] Boyer | Infectious transcripts and cDNA clones of RNA viruses[END_REF], and the toxicity of some viral proteins in Escherichia coli, resulting in spontaneous deletions of some clones in bacterial cells. The most toxic proteins appear to be P3 and CI encoded in the central region of the genome [START_REF] Jakab | Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of potato virus Y[END_REF][START_REF] Ali | An infectious full-length cDNA clone of potato virus YNTN-NW, a recently reported strain of PVY that causes potato tuber necrotic ringspot disease[END_REF]. In order to overcome these difficulties, several techniques have been used, including cloning the genome in two halves that have to be religated in vitro before inoculation [START_REF] Jakab | Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of potato virus Y[END_REF], adding introns in the cDNA to prevent expression in E. coli [START_REF] Johansen | Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo[END_REF], or removing cryptic prokaryotic promoter elements in the P3 coding region by site-directed mutagenesis [START_REF] Ali | An infectious full-length cDNA clone of potato virus YNTN-NW, a recently reported strain of PVY that causes potato tuber necrotic ringspot disease[END_REF]. One drawback of these techniques, besides the fact that they are time-consuming, is the risk of adding unwanted mutations in extensively manipulated clones, resulting in non-infectivity or impaired biological properties. To limit theses drawbacks, a simple protocol involving a very limited number of cloning steps and proven to be very efficient was developed.

Purified virions of strains WMV-FMF00-LL2 and WMV-FMF00-LL1 [START_REF] Desbiez | Serological and molecular variability of watermelon mosaic virus (genus Potyvirus)[END_REF] obtained with standard protocols applied in the laboratory [START_REF] Lecoq | Specificity of the helper-component-mediated aphid transmission of three potyviruses infecting muskmelon[END_REF] immediately before use (in the case of FMF00-LL2) or kept in glycerol (1:1 vol:vol) at -20°C for 2 years were used for the production of viral RNA. The virus particles were precipitated by ultracentrifugation for 2 h at 130000 g in a Beckman LE-80 ultracentrifuge with a 50.2Ti rotor, and resuspended in 0.02 M potassium phosphate buffer at pH 7.4. One µg of purified virus was subjected to a Proteinase K treatment for 40 min at 37°C (100 µM Proteinase K in buffer Tris 10mM, EDTA 5 mM, SDS 0.5% pH 8), then to one phenol extraction followed by a phenol: chloroform: isoamylate (25:24:1) extraction, ethanol precipitation and resuspension in 20 µl RNAse-free water. Viral genomes were amplified in four overlapping fragments of 1.9 to 3.5 kb using specific primers with at least a 20-base overlap (Fig. 1, Table 1). The primers corresponding to the 5' and 3' extremities of the genome contained an extra 20-to 30-base tail corresponding to the extremities of the vector (see below). Reverse transcription reactions were performed on 2µl RNA, using 2.5 U AMV reverse transcriptase (Promega, France) in the manufacturer's buffer, 0.5 mM each dNTP, and 0.5 µM reverse primers WMV-A-RV, C-RV, E-RV and F-RV (Table 1). The reactions were performed for 1 h at 42°C. Two µl of each cDNA were used for PCR amplification using 1-2 U Pfu DNA polymerase (Promega, France) with 0.2 mM of each dNTP and 0.5 µM of each primer. PCR reactions were performed as follows: 5 min at 95°C, followed by 35 cycles of 45 s at 95°C, 45 s at 50°C and 5 min at 72°C. PCR fragments of the expected sizes were excised from 1% agarose gel under UV light and purified using standard protocols.

For WMV, numerous attempts had shown that viral clones were highly unstable in E. coli (Desbiez, unpublished), and the insertion of introns was chosen as a way to improve the stability of bacterial clones as used by [START_REF] Johansen | Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo[END_REF] for pea seedborne mosaic virus. Introns IV (189 bp) of the ST-LS1 gene from Solanum tuberosum ("Intron 1" in this work) and Intron 2 (221pb) of the NiR gene from Phaseolus vulgaris [START_REF] Johansen | Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo[END_REF] were amplified from an intron-containing infectious clone of potato virus Y (PVY) SON41p [START_REF] Moury | Mutations in Potato virus Y genome-linked protein determine virulence toward recessive resistances in Capsicum annuum and Lycopersicon hirsutum[END_REF], using primers "WMV-B-FW + WMV-B-RV" and "WMV-D-FW + WMV-D-RV" containing a 30-base tail complementary to the genome of WMV at positions 3541 and 5397 in the P3 and CI coding regions respectively (Table 1). Amplified fragments B and D (corresponding to introns 1 and 2, respectively) were fused to fragments C and E, respectively, by fusion PCR (Fig. 1): 0.2 µl of fragments "B and C" and "D and , respectively, were used in a mix containing 1-2 U Pfu in the manufacturer's buffer, 0.2 mM of each dNTP, 0.5 µM each of primers "WMV-B-FW + WMV-C-RV" and "WMV-D-FW + WMV-E-RV". The amplification conditions were as above with elongation times of 4 min. Fragments of the expected size were excised from agarose gels and purified.

To prepare the vector, a construct containing the full-length clone of PVY SON41p in pAGUS1 [START_REF] Johansen | Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo[END_REF][START_REF] Moury | Mutations in Potato virus Y genome-linked protein determine virulence toward recessive resistances in Capsicum annuum and Lycopersicon hirsutum[END_REF], containing the 2µ yeast replication origin and Trp-1 promoter and gene as a selectable marker [START_REF] Sikorski | A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae[END_REF] was digested with PflFI and NcoI in order to excise almost completely the PVY insert. The 5.5 kb fragment corresponding to the vector was excised from an agarose gel and purified.

The diploid Saccharomyces cerevisiae strain YPH501 (YPH500xYPH499) (MATa/MATα ura3-52/ura3-52 lys2-801_amber/lys2-801_amber ade2-101_ochre/ade2-101_ochre trp1-Δ63/trp1-Δ63 his3-Δ200/his3-Δ200 leu2-Δ1/leu2-Δ1), auxotrophic for tryptophan, was used [START_REF] Sikorski | A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae[END_REF]. Yeast cultures were grown at 28°C in YPD medium (1% yeast extract, 2% peptone, 2% Dglucose) prior to transformation and in CAU selective medium (0.1% casamino acid containing no tryptophan, 0.67% yeast nitrogen base without amino acids, 0.0025% adenine sulfate, 0.0025% uracil, 2% D-glucose) after transformation. Preparation of competent cells and transformation were performed using a protocol derived from [START_REF] Klebe | A general method for polyethyleneglycol-induced genetic transformation of bacteria and yeast[END_REF]. Briefly, yeasts were grown in 500 ml YPD medium at 30°C with agitation, until absorbance at 600nm reached 0.6. The cells were centrifuged for 3 min at room temperature and resuspended in 250 ml of SBEG (sorbitol 1 M, bicine pH 8.5 10 mM, ethylene glycol 3%) After a second centrifugation, the cells were resuspended in 5 ml SBEG, and aliquots of 100 µl were stored at -80°C until use. For transformation, 100 µl of competent yeast cells were mixed with 7 µl of sonicated heatdenaturated salmon sperm DNA (10 mg/ml) as a carrier and 15 µl of vector+insert DNA containing approximately the same amount of the different insert fragments (about 100 ng), with a vector:insert ratio of 1:3. Eight hundred µl of PEG4000 (polyethylene glycol 40%, bicine 10 mM, pH 8.35) were added, and the cells were incubated for one hour at 30°C with gentle agitation. The cells were collected by centrifugation for 5 min at 2600 g, and the pellet was resuspended in 1 ml NB buffer (NaCl 0.15 M, bicine 10mM, pH 8.35). After 5 min of centrifugation at 2600 g, 900 µl of the supernatant was discarded; the cells were resuspended in the remaining 100 µl, plated on CAU solid medium (containing 2% bacto-agar) and grown at 30°C for at least 48 h. Yeast colonies (3 to 4 colonies per transformation) were inoculated in 3 ml of liquid CAU medium and grown overnight at 28°C. Plasmid DNA was isolated according to [START_REF] Hoffman | A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli[END_REF]. Briefly, the cells were collected by centrifugation and resuspended in 200 µl of a buffer containing 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8, 1 mM EDTA. Two hundred µl of phenol:chloroform:isoamylic alcohol (50:49:1) and 0.3 g of 0.45 mm acid-washed glass beads (Sigma) were added, and the suspension was vigorously shaken for 5 min. The DNA was recovered by ethanol precipitation, resuspended in 10 µl of de-ionized water, desalted on a Millipore MF TM 0.025µ membrane filter and used for transformation of electrocompetent E. coli DH5 according to standard protocols. Bacterial plasmid DNA was extracted by alkaline lysis and the screening was performed by restriction enzyme analysis. The plasmids were inoculated with a HandGun according to [START_REF] Gal-On | Particle bombardment drastically increases the infectivity of cloned DNA of zucchini yellow mosaic potyvirus[END_REF] to 10 susceptible zucchini squash F1 Diamant plantlets at the cotyledon stage. Six independent clones were pooled for the first bombardment, using approximately 30 ng DNA per clone, in order to determine if there was at least an infectious clone among them. Three and 8 plants were positive after inoculation with FMF00-LL2 and FMF00-LL1, respectively, as defined by symptom observation and serological (ELISA) tests with a WMV polyclonal antiserum. In a second step, each clone was inoculated individually to 10 zucchini plantlets, and symptoms were checked visually two weeks after inoculation. Viral infection was confirmed by ELISA tests. Two and 4 out of 6 clones each were found to be infectious for FMF00-LL2 and FMF00-LL1 respectively. The presence of the expected viral strain was checked in symptomatic plants by RNA [START_REF] Desbiez | Serological and molecular variability of watermelon mosaic virus (genus Potyvirus)[END_REF]. Symptoms observed after bombardment of the infectious clones and subsequent mechanical inoculations were indistinguishable from those induced by the wild-type isolates. There was no delay in the appearance of symptoms after mechanical inoculation from zucchini plants infected with the clones compared to those infected by wild-type viruses (data not shown). The host range of the infectious clones appeared similar to that of the natural isolates (data not shown), with FMF00-LL1 being systemic on Ranunculus sardous but not on Chenopodium quinoa, and FMF00-LL2 being systemic on C. quinoa but not on R. sardous [START_REF] Lecoq | Search for factors involved in the rapid shift in watermelon mosaic virus (WMV) populations in south-eastern France[END_REF]. For ZYMV and PRSV, since several infectious intronless clones have already been obtained and are satisfactorily stable [START_REF] Gal-On | Infectious in vitro RNA transcripts derived from cloned cDNA of the cucurbit potyvirus, zucchini yellow mosaic virus[END_REF][START_REF] Lin | Construction of in vitro and in vivo infectious transcripts of a Taiwan strain of Zucchini yellow mosaic virus[END_REF][START_REF] Chiang | Infectivity assays of in vitro and in vivo transcripts of papaya ringspot potyvirus[END_REF][START_REF] Chen | A single amino acid of NIaPro of Papaya ringspot virus determines host specificity for infection of papaya[END_REF], it could be expected that clones of these viruses would not require introns. Consequently, a simplified cloning strategy was used. RNA was extracted from purified virions of two reference isolates from France, ZYMV-E15 [START_REF] Lecoq | Strains of zucchini yellow mosaic virus in muskmelon (Cucumis melo L.)[END_REF] and PRSV-E2 [START_REF] Lecoq | Mise en évidence du virus de la mosaïque de la pastèque type 1 (WMV-1) dans le Sud-Est de la France[END_REF], stored in 50% glycerol at -20°C for more than 15 years, as for WMV. Four overlapping fragments of about 2.5 kb each were amplified by RT-PCR as above, purified on gel and used for yeast transformation with the same vector as for WMV. Four yeast colonies for each virus were grown in liquid CAU medium; DNA was extracted as for WMV and used for transformation of E. coli. Since the complete sequences of ZYMV-E15 and PRSV-E2 had not been established beforehand and their restriction profiles were not known, bacterial plasmid DNA was digested with EcoRI. This enzyme was chosen because 3 to 6 EcoRI restriction sites are usually present in the full-length sequences of ZYMV and PRSV available in databases (data not shown). Clones displaying a total size of about 15 kb were used for bombardment as for WMV. In a first assay, a pool of 3 clones for each virus was used and was shown to be infectious by symptom observation and serological tests. After separate bombardments, 2 and 1 clones were infectious for ZYMV and PRSV, respectively. The symptoms induced by the cloned cDNAs were indistinguishable from those of wild-type strains. Pathotypes of cloned ZYMV-E15 on melon lines carrying the Fn or Zym genes [START_REF] Lecoq | Strains of zucchini yellow mosaic virus in muskmelon (Cucumis melo L.)[END_REF] were identical to those of wild-type ZYMV-E15 (data not shown). In both cases, the whole cloning process from RNA purification to plant bombardment took about 10 days. The PCR steps were performed from previously obtained purified viruses that were available, which may have improved the PCR efficiency due to the purity of the RNA used, as well as the infectivity of the clones obtained since only RNAs that had been able to be encapsidated were involved in the amplification and cloning processes. The PCR steps worked satisfactorily with total RNA of infected plants (data not shown), but the percentage of fragments containing highly deleterious or lethal mutations in this case is not known. Using immunocapture [START_REF] Wetzel | A highly sensitive immunocapture polymerase chain reaction method for plum pox virus detection[END_REF] before the RT-PCR may allow selection of only encapsidated RNA in the amplification procedure without requiring a virus purification step. As a whole, this cloning strategy did not involve any exceptional techniques, but it was surprisingly rapid and efficient. The insertion of introns has already been used for stabilizing cDNA clones of several potyviruses [START_REF] Johansen | Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo[END_REF]Lopez-Moya et al., 2002[START_REF] Moury | Mutations in Potato virus Y genome-linked protein determine virulence toward recessive resistances in Capsicum annuum and Lycopersicon hirsutum[END_REF]) even if they were inserted step by step using ligation in suitable restriction sites. Homologous recombination in yeast rather than ligation in bacteria has also proved useful for generating recombinant clones of plant viruses [START_REF] Fernandez-Delmond | A novel strategy for creating recombinant infectious RNA virus genomes[END_REF][START_REF] Liang | Site-Directed Mutagenesis and Generation of Chimeric Viruses by Homologous Recombination in Yeast to Facilitate Analysis of Plant-Virus Interactions[END_REF] Infectious clones of apple chlorotic leaf spot virus have been recently obtained by one-step cloning of the 7.5 kb genome in an agroinfiltration vector [START_REF] Youssef | Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs : Apple chlorotic leaf spot virus as a case study[END_REF]. Yeast transformation with 4 insert fragments (2.5 kb each) and 1 vector yielded good results, with 30 to 50 yeast colonies obtained per transformation. In another attempt, transformation with 5 insert fragments of 1.5 to 2.5 kb resulted in about 20 colonies (data not shown). On the other hand, a first attempt of cloning for WMV with 1 vector, 4 virus fragments (2.5 kb each) and 2 intron fragments (200 pb each) did not yield any colonies. It is not known whether this failure was related to the number of recombining fragments, or to the small size of the introns. An argument against the first hypothesis is the fact that a complete mycoplasma [START_REF] Gibson | One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome[END_REF]. In this work, fusion PCR was a way to overcome the problem. The percentage of infectious clones obtained among the ones generated was from 30% to 60% in the conditions used. The actual percentage may be slightly higher, the "non-infectivity" could be related in some cases to non-optimal concentration of the plasmid DNA or inoculation conditions. However an infectivity of about 50% probably reflects the ratio of infectious to non-infectious genomes in the original virus population, since potyviruses, with a genome of 10 kb, have a mutation rate in the order of magnitude of 10 -4 mutations/genome/replication cycle [START_REF] Sanjuan | Viral mutation rates[END_REF], and more than half of these mutations are lethal or significantly deleterious [START_REF] Carrasco | Distribution of fitness and virulence effects caused by single-nucleotide substitutions in Tobacco etch virus[END_REF]. Testing a sufficient number of clones (3 to 6 clones here) can overcome the problem. In classic cloning procedures, this would mean handling a large and sometimes exponentially-growing number of constructs during the cloning process, but with this protocol involving a very limited number of steps, the number of clones that can be tested is not a limiting factor. This work very likely represents the first time that infectious potyvirus cDNA clones are obtained repeatedly in less than 2 weeks starting almost from scratch. This simple technique opens very promising perspectives to obtain routinely infectious clones for other potyviruses.

  Figure 1: Cloning strategy for WMV (a) Cloning of WMV genome using homologous recombination in yeast, with 4 genomic fragments overlapping the complete genome (fragments A, C, E, F) and 2 introns (fragments B and D), in a shuttle vector containing a 35S promoter and nos terminator (b) Introns B and D were fused to fragments C and E respectively by fusion PCR before cloning

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : Journal of VirologicalMethods, 2012Methods, , 183, 94-97, http://dx.doi.org/10.1016Methods, /j.jviromet.2012.03.035. .03.035.

	Manuscrit d'auteur / Author manuscript
	Manuscrit d'auteur / Author manuscript
	Manuscrit d'auteur / Author manuscript	2

. A simple, rapid and efficient way to obtain infectious clones of potyviruses. Journal of Virological Methods, 183 (1), 94-97. DOI : 10.1016/j.jviromet.2012.03.035

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : Journal of VirologicalMethods, 2012Methods, , 183, 94-97, http://dx.doi.org/10.1016Methods, /j.jviromet.2012.03.035. .03.035.

	E"
	Manuscrit d'auteur / Author manuscript
	Manuscrit d'auteur / Author manuscript
	Manuscrit d'auteur / Author manuscript	3

Comment citer ce document : Desbiez, C. (Auteur de correspondance), Chandeysson, C., Lecoq, H., Moury, B. (2012). A simple, rapid and efficient way to obtain infectious clones of potyviruses. Journal of Virological Methods, 183 (1), 94-97. DOI : 10.1016/j.jviromet.2012.03.035

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : Journal of VirologicalMethods, 2012Methods, , 183, 94-97, http://dx.doi.org/10.1016Methods, /j.jviromet.2012.03.035. .03.035.

	according to standard protocols
	Manuscrit d'auteur / Author manuscript
	Manuscrit d'auteur / Author manuscript
	Manuscrit d'auteur / Author manuscript	4

extraction, RT-PCR and partial sequencing Comment citer ce document : Desbiez, C. (Auteur de correspondance), Chandeysson, C., Lecoq, H., Moury, B. (2012). A simple, rapid and efficient way to obtain infectious clones of potyviruses. Journal of Virological Methods, 183 (1), 94-97. DOI : 10.1016/j.jviromet.2012.03.035

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : Journal of VirologicalMethods, 2012Methods, , 183, 94-97, http://dx.doi.org/10.1016Methods, /j.jviromet.2012.03.035. .03.035.

		genome was recently obtained by homologous recombination in yeast of 25 genomic fragments
	Manuscrit d'auteur / Author manuscript
	Manuscrit d'auteur / Author manuscript
	Manuscrit d'auteur / Author manuscript	5

Comment citer ce document : Desbiez, C. (Auteur de correspondance), Chandeysson, C., Lecoq, H., Moury, B. (2012). A simple, rapid and efficient way to obtain infectious clones of potyviruses. Journal of Virological Methods, 183 (1), 94-97. DOI : 10.1016/j.jviromet.2012.03.035

Comment citer ce document : Desbiez, C. (Auteur de correspondance), Chandeysson, C., Lecoq, H., [START_REF] Desbiez | A simple, rapid and efficient way to obtain infectious clones of potyviruses[END_REF] Underlined sequences correspond to the extremities of the introns. Sequences in italics correspond to the extremities of the vector.