Laurence Svanella-Dumas

Iason Tsarmpopoulos

Armelle Marais

Chantal Faure

Sebastien Theil

Miroslav Glasa

Lukas Predajna

Jonathan Gaudin

Sixing Tian

Laetitia Porcher

Sébastien Theil

Laëtitia Porcher

Pascal Gentit

Milena Leite De Oliveira

Renate Krause-Sakate

Thierry Candresse
email: thierry.candresse@inrae.fr

Molecular and biological characterization of novel and known Sequiviridae members infecting lettuce

Keywords: Lettuce, France, Brazil, lettuce mottle virus, High-throughput sequencing, etiology

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.

INTRODUCTION

Lettuce (Lactuca sativa L.) is the most important annual leafy vegetable crop. It is primarily consumed fresh and is considered an important dietary vegetable worldwide. It belongs to the Asteraceae, the largest family of flowering plants. Cultivated lettuce was first documented on the walls of Egyptian tombs 4500 years ago and was soon cultivated in most parts of Europe and, later, in other parts of the world [START_REF] De Vries | Origin and domestication of Lactuca sativa L[END_REF]. The main producer of lettuce is Asia, especially India and China which, according to FAO in 2019, ranks first in annual production, followed by North and Central America, and Europe. In the European Union, Spain, Italy, Germany and France are the major lettuce-producing countries, with 74% of the total European lettuce production at almost 1.75 million tons in 2017 (Le Fustec 2020). In France, lettuce is grown year around in open fields, under plastic houses or glasshouses, with a total acreage of 8,300 hectares corresponding to about 500 million plants annually [START_REF] Fustec | Laitues d'hiver 2019-2020: une fin de campagne marquée par l'impact de la crise sanitaire sur la demande et les dernières récoltes[END_REF].

A number of pests and pathogens affect lettuce crops, including viruses belonging to the genera Alfamovirus, Crinivirus, Cucumovirus, Cytorhabdovirus, Fabavirus, Ophiovirus, Polerovirus, Potyvirus, Sequivirus, Tombuvirus, Orthotospovirus and Varicosavirus (Ciuffo et al. 2016;Ephytia 2019;[START_REF] Jadao | Further characterization of two sequiviruses infecting lettuce and development of specific RT-PCR primers[END_REF][START_REF] Lebeda | Wild Lactuca species, their genetic diversity, resistance to diseases and pests, and exploitation in lettuce breeding[END_REF][START_REF] Moreno | Virus diseases in lettuce in the Mediterranean basin[END_REF]Pavan et al. 2008). Many of these viruses cause epidemic diseases and considerable yield losses.

Thanks to developments in high throughput sequencing (HTS) combined with specific bioinformatic tools [START_REF] Lefebvre | The VirAnnot pipeline: a resource for automated viral diversity estimation and operational taxonomy units assignation for virome sequencing data[END_REF][START_REF] Villamor | High throughput sequencing for plant virus detection and discovery[END_REF], numerous plant viruses have been recently characterized, allowing major advances in the study of the etiology of viral diseases in perennial or annual crops [START_REF] Wu | Identification of viruses and viroids by next-generation sequencing and homology-dependent and homology-independent algorithms[END_REF][START_REF] Maliogka | Recent advances on detection and characterization of fruit tree viruses using high-throughput sequencing technologies[END_REF][START_REF] Maree | Application of HTS for routine plant virus diagnostics: state of the art and challenges[END_REF]). However, in many cases, past the identification and genome characterization of novel viral agents, the challenge is to demonstrate their potential implication in diseases and to provide information about their biology, allowing to evaluate the risks these novel agents may pose (Massart et al. 2017).

In recent years, several novel viruses have been characterized in lettuce crops [START_REF] Ciuffo | Molecular identification and biogical characterization of a new potyvirus in lettuce[END_REF][START_REF] Svanella-Dumas | Complete genome sequence of lettuce chordovirus 1 isolated from cultivated lettuce in France[END_REF][START_REF] Verbeek | Lettuce necrotic leaf curl virus, a new plant virus infecting lettuce and a proposed member of the genus Torradovirus[END_REF], while the main viruses infecting lettuce in Europe or in Central America have been respectively reviewed in [START_REF] Moreno | Virus diseases in lettuce in the Mediterranean basin[END_REF] and [START_REF] Pavan | Virus diseases of lettuce in Brazil[END_REF]. Lettuce mosaic virus (LMV, Potyvirus) is probably the most important virus of lettuce worldwide [START_REF] Pavan | Virus diseases of lettuce in Brazil[END_REF]. LMV causes typical mosaic symptoms that may occasionally turn necrotic. However, some other lettuce viruses can cause almost indistinguishable mosaic symptoms, such as lettuce mottle virus (LeMoV) and dandelion yellow mosaic virus (DaYMV), both of them from the genus Sequivirus in the family Secoviridae [START_REF] Jadao | Further characterization of two sequiviruses infecting lettuce and development of specific RT-PCR primers[END_REF]. DaYMV was the first sequivirus reported in lettuce and in plants of dandelion (Taraxacum officinale) in different European countries [START_REF] Kassanis | A virus attacking lettuce and dandelion[END_REF][START_REF] Kassanis | Studies on dandelion yellow mosaic and other virus diseases of lettuce[END_REF][START_REF] Bos | Further characterization of dandelion yellow mosaic virus from lettuce and dandelion[END_REF][START_REF] Jadao | Further characterization of two sequiviruses infecting lettuce and development of specific RT-PCR primers[END_REF]). LeMoV, differing from DaYMV in host range, is a distinct sequivirus infecting lettuce in Brazil and in Chile [START_REF] Krause-Sakate | First report of a lettuce-infecting sequivirus in Chile[END_REF][START_REF] Jadao | Further characterization of two sequiviruses infecting lettuce and development of specific RT-PCR primers[END_REF]. In Brazil, and in particular in Sao Paulo State, LeMoV was in the early 2000s the most frequent virus in lettuce crops [START_REF] De Marchi | Levantamento revela a predominância do Lettuce mottle virus em três regiões produtoras de alface no Estado de São Paulo[END_REF][START_REF] Jadao | Further characterization of two sequiviruses infecting lettuce and development of specific RT-PCR primers[END_REF] but it has so far never been reported outside of South America. For both DaYMV and LeMoV, only very short, partial genome sequences are available [START_REF] Jadao | Further characterization of two sequiviruses infecting lettuce and development of specific RT-PCR primers[END_REF] and there is very little information available on their genetic variability.

We report here the biological and molecular characterization by HTS of several secoviruses from wild and cultivated lettuce in France and Brazil and from dandelion in Slovakia. Besides providing the first complete genomic sequence for LeMoV and near-complete genome sequences for DaYMV, the results presented here describe a novel Sequivirus and a novel Waikavirus detected in French cultivated lettuces.

MATERIALS AND METHODS

Plant samples and virus isolates

To identify the etiological agent(s) involved, three cultivated lettuces (Lactuca sativa L.) presenting systemic mosaic symptoms with some necrotic points were analyzed by HTS. The first one (cv. Shangore, JG1) was collected in 2014 near Sainte-Livrade (southwestern France) and the second one (cv. Guétary, JG2) came from an organic plot under a plastic Two isolates of dandelion yellow mosaic virus (DaYMV) in mixed infection, RNA24P-1 and RNA24P-2, were identified by HTS from a dandelion plant collected in 2018 at the border of a cultivated field near Bratislava, Slovakia.

Host range study and sap inoculation of viruses

In order to characterize the novel viruses identified, leaf pieces from the original JG1 plant were ground 1:3 (wt/vol) in 0.05M potassium phosphate buffer pH 8.0. Activated charcoal and carborundum (400 mesh) were added (100mg/ml homogenate) and the mixture was used for sap-inoculation of plants of L. sativa (cv. Trocadero) or of ten plant species belonging to the families Asteraceae, Solanaceae, Chenopodiaceae and Amaranthaceae. The LeMoV isolate identified in the JG2 plant was sap-transmitted to lettuce (cv. Trocadéro), Nicotiana benthamiana and Gomphrena globosa plants using the buffer described in [START_REF] Jadao | Further characterization of two sequiviruses infecting lettuce and development of specific RT-PCR primers[END_REF].

The host range study was repeated at least twice for each viral isolate. All plants were kept under greenhouse conditions and monitored for symptoms development up to one-month post-inoculation.

The presence of the viruses in upper non-inoculated leaves of the test plants was evaluated using specific RT-PCR assays for each virus using total nucleic acids (TNA) extracted from fresh leaves according to protocol 1 of [START_REF] Foissac | Polyvalent degenerate oligonucleotides reverse transcription-polymerase chain reaction: a polyvalent detection and characterization tool for trichoviruses, capilloviruses, and foveaviruses[END_REF]. The sequences of the various amplification primers used [New waika-F / New waika-R (for the new waikavirus) and NSeco-varA-F / NSeco-varA-R or NSeco-varB-F / NSeco-varB-R (that discriminate between the two variants of the new sequivirus] are provided in Supplementary Table S1. These primer pairs target fragments of respectively 385, 445 or 546 nucleotides (nt) in the viral RNA-dependent RNA polymerase (RdRp) gene of each virus. The presence of LeMoV was similarly checked using primer pair LeMoV3 [START_REF] Jadao | Further characterization of two sequiviruses infecting lettuce and development of specific RT-PCR primers[END_REF]) and LeMoV4-multi (Supplementary Table S1) which is adapted from [START_REF] Jadao | Further characterization of two sequiviruses infecting lettuce and development of specific RT-PCR primers[END_REF] so as to improve inclusiveness and to allow amplification of isolates JG2, P22, SP described here and of previously analyzed Brazilian LeMoV isolates (DQ675190-91). This primer pair amplifies a 667 nt fragment of the RdRp gene.

For each RT-PCR assay, cDNA was first synthesized from 5µl of TNA using dT18 and pdN6 primers and the Revertaid Reverse Transcriptase (200U/µl) according to the manufacturer recommendations (Thermo Fisher Scientific, Illkirch, France). In a second step, the PCR was carried out using 3 µl of cDNA in a 25-µl reaction volume using 1 U of DyNAzymeII™ DNA polymerase in accordance with the manufacturer recommendations (Thermo Fisher Scientific). The annealing temperature and duration of the 72°C extension step are provided in Supplementary Table S1. PCR products were analyzed by non-denaturing 1% agarose gel electrophoresis with ethidium bromide staining and, if needed, directly sequenced (Eurofins Genomics, Ebersberg bei München, Germany) to verify the specificity of the amplification reaction.

HTS analysis, genome assembly and completion of the genome of the various viral isolates

Double-stranded RNAs (dsRNAs) were purified from fresh symptomatic leaves of the original JG1, JG2, P22 and LIB3 plants as described [START_REF] Marais | Viral double-stranded RNAs (dsRNAs) from plants: alternative nucleic acid substrates for high-throughput sequencing[END_REF]. Purified dsRNAs were submited to a random amplification [START_REF] Marais | Viral double-stranded RNAs (dsRNAs) from plants: alternative nucleic acid substrates for high-throughput sequencing[END_REF]) and sequenced on Illumina platforms (2×250 or 2x150 nt). Total RNAs were purified from N. benthamiana leaves infected by the LeMoV-JG2 isolate using the protocol of Chang et al. (1983) and sequenced without ribodepletion an Illumina HiSeq2000 (2×150 nt). Additionally, total RNAs were extracted from fully developed dandelion leaves as mentioned in Tomasechova et al. (2020) and sequenced on an Illumina MiSeq (2×250 nt). Total RNAs extracted from the 21-015C lettuce were similarly sequenced on the Illumina iSeq100 and MiSeq platforms (2x150nt).

Following demultiplexing and quality trimming, HTS reads from the various samples were analyzed using CLC Genomics Workbench 8 and later versions (https://www.qiagenbioinformatics.com) and the VirAnnot pipeline [START_REF] Lefebvre | The VirAnnot pipeline: a resource for automated viral diversity estimation and operational taxonomy units assignation for virome sequencing data[END_REF]. De novo assembled contigs were annotated by BlastN and BlastX analyses against the GenBank database using a 10 -3 e-value cut-off. Viral contigs were further extended by several rounds of mapping of residual reads and by integration of smaller contigs to yield scaffold covering most of the viral genomes.

The 5'end of viral genomes was determined using a 5' RACE kit (Takara Bio Europe/Clontech, Saint-Germain-en-Laye, France) and specific reverse primers designed on the reconstructed scaffold of each virus (Supplementary Table S1). To complete the 3'end of the genomes of the sequiviruses, that do not contain a poly-A tail, TNA were extracted and polyadenylated (Ambion/ThermoFisher Scientific, Illkirch, France). The polyadenylated RNAs were then submitted to a reverse transcription using LD-polyT as a reverse primer and the genome 3'end was then amplified by PCR using a forward-specific internal primer designed from the genome scaffold sequenced contig (Supplementary Table S1) and the LDprime primer, following the protocol described by Youssef et al. (2011). The same procedure was used to determine the Waikavirus 3' genome end, only omitting the polyadenylation step since waikavirus genomes are polyadenylated.

Internal gaps and regions of low coverage were determined or confirmed by direct sequencing of RT-PCR fragments obtained using internal primers designed from the sequences of contigs. For LeMoV-JG2, the only gap identified was in the 3' part of the genome, in a C-rich region with low coverage. Two combinations of primers (LeMoV-3Race1 / LeMoV-gap-R1

and LeMoV-3Race1 / LeMoV-gap-R2) (Supplementary Table S1), amplifying PCR products of respectively 199 and 573 nt were used to confirm the genome sequence in this short region.

The nearly genomic sequence of the LeMoV isolate P22 was completed after designing six pairs of primers (Supplementary Table S1) allowing to close six small internal gaps in the HTS scaffold.

All amplified fragments were visualized on non-denaturing agarose gels and directly submitted to Sanger sequencing on both strands (Eurofins Genomics) or, when needed, sequenced after cloning in the pGEM-T Easy vector (Promega, Charbonnières-Les Bains, France).

The nearly complete genome of the LeMoV isolate SP was obtained by RT-PCR-based genome walking, using total RNAs extracted from infected C. quinoa leaves with the Total RNA Purification Kit (Norgen Biotek, Thorold, Canada). The primers used (Supplementary Table S1) were designed based on the genome sequence of the LeMoV P22 isolate. The onestep RT-PCR reactions were performed using 2.5 µl of RNA, 1 µM of each primer and 0.03 units of Avian myeloblastosis virus reverse transcriptase (AMV RT, Promega, Charbonnièresles-Bains, France) in a final volume of 25 µl of 1x PCR master mix (Fermentas, Vilnius, Lithuania). The annealing temperature for each primer pair is provided in Supplementary Table S1. PCR products were analyzed as described above, purified from agarose gels using the QIAquick Gel Extraction Kit (Qiagen, Courtaboeuf, France) and sequenced using an ABI 3730 DNA Analyser (Applied Biosystems, Illkirch, France) using a BigDye Terminator v3.1

Cycle Sequencing kit (Applied Biosystems).

Sequence comparisons and phylogenetic analyses

Multiple nucleotide (nt) or deduced amino acid (aa) sequence alignments were performed using ClustalW [START_REF] Thompson | CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap, penalties and weight matrix choice[END_REF]) implemented in MEGA 7 [START_REF] Kumar | MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets[END_REF]. Strict identity nt or aa distances were calculated from these alignments and neighbor-joining phylogenetic trees were reconstructed using Mega 7 with bootstrap analysis (500 replicates)

to evaluate branch validity.

RESULTS

Viruses detected by HTS in the French JG1 and JG2 lettuces

During the spring of 2014 and 2015, two symptomatic lettuces (JG1, cv. Shangore and JG2 cv. Guétary) showing systemic mosaic symptoms were observed during surveys. Both varieties are resistant to lettuce downy mildew (Bremia lactucae Regel) but only JG2 is resistant to lettuce mosaic virus (LMV) and to the lettuce aphid, Nasonovia ribisnigri. The symptoms on the JG2 lettuce included some necrotic points (Fig. 1A) that were much less present on the JG1 plant. Symptomatic leaves of both plants were used to mechanically inoculate lettuce plants (cv. Trocadéro), which developed systemic mosaic symptoms, confirming suspicions of viral infection. In order to identify the virus(es) involved, dsRNAs were purified from 0.75g of fresh leaves from the original JG1 and JG2 plants and analyzed by HTS. Following demultiplexing and quality trimming, the reads were assembled into contigs that were annotated by BlastN and BlastX analyses, providing evidence for a mixed infection by several agents for the JG1 lettuce and for a single infection in JG2 (Table 1).

For JG1, the contig integrating the most reads (26,980)

Genome organization of the Waikavirus identified in the JG1 lettuce

Following further rounds of reads assembly and aggregation of shorter contigs, a nearly complete genome scaffold of 11,852 nt was assembled for the tentative Waikavirus identified in the JG1 plant. The missing terminal genome ends were determined by 5'-RACE and 3' LD-polyT / LD-prime PCR (Youssef et al. 2011) using the purified dsRNAs from the original plant as a template and specific primers designed from the scaffold sequence (Supplementary Table S1). The completed genome is 11,929 nt in length, excluding the 3'poly (A) tail (Fig. 2A) and has been deposited in GenBank under the MT348710 accession number. The genome organization is similar to that of other waikaviruses, with a single large open reading frame (ORF) of 10,389 nt encoding a polyprotein of 3,463 aa (388.5 kDa). The 5' and 3' untranslated regions (UTRs) are respectively 463 nt and 1,078 nt long. The presence of additional potential ORFs has been reported for some waikaviruses [START_REF] Shen | Nucleotide sequence and genomic organization of rice tungro spherical virus[END_REF][START_REF] Firth | Bioinformatic analysis suggests that a conserved ORF in the waikaviruses encodes an overlapping gene[END_REF]). The predicted short overlapping ORFX was identified starting at genome position BVCV and variable conservation levels with other genus members (Supplementary Figure 1).

On the other hand, the long 3' UTR of the JG1 waikavirus does not contain the small ORF2 and ORF3 described in rice tungro spherical virus (RTSV) by [START_REF] Thole | Rice tungro spherical virus: nucleotide sequence of the 3' genomic half and studies on the two small 3' open reading frames[END_REF]. In waikaviruses and sequiviruses, the single large polyprotein encodes functional proteins released by post-translational cleavages mediated by the viral protease (Pro). From the Nterminal polyprotein end, these are a P1 protein of unknown function, the three coat protein subunits (CP1, CP2, and CP3), an NTP-binding helicase (NTB), the 3C-like protease and an

RdRp. The polyprotein cleavage sites have been experimentally identified for maize chlorotic dwarf virus (MCDV) and RTSV [START_REF] Reddick | Nucleotide sequence and taxonomy of maize chlorotic dwarf virus within the family Sequiviridae[END_REF][START_REF] Shen | Nucleotide sequence and genomic organization of rice tungro spherical virus[END_REF] "YGDD" (aa 3258 to 3261) motifs [START_REF] Reddick | Nucleotide sequence and taxonomy of maize chlorotic dwarf virus within the family Sequiviridae[END_REF][START_REF] Pavan | Virus diseases of lettuce in Brazil[END_REF]) characterizing the RdRp domain.

Genome organization of the sequivirus isolates identified in the JG1 lettuce

As indicated above, two long scaffolds representing sequivirus isolates could be reconstructed for the JG1 plants, named here JG1-A (8,961 nt) and JG1-B (9,158 nt). The whole genome of the JG1-B was completed by 5'-RACE and 3' LD-polyT / LD-prime PCR as described above for the Waikavirus sequence, with the only exception that the genome 3' end was determined after polyadenylation since sequiviruses do not possess a 3' polyA. No specific efforts were made to complete the JG1-A genome sequence. The JG1-B complete genome is 10,183 nt long and has been deposited in GenBank, together with the JG1-A contig sequence (MT348706 and MT348705, respectively). As expected, the JG1-B genome harbors a single large ORF of 9,054 nt encoding a predicted polyprotein of 3,018 aa (337.5 kDa), bordered by 5' and 3' UTRs of 150 nt and 980 nt, respectively (Fig. 2B). Conserved protein motifs were identified at the expected locations in the JG1-B polyprotein. This includes the A "GSPGVGKS" (aa 1430 to 1437), B "DD" (aa 1481 to 1482) and C "KGRTFSSKYIFSTTN"

(aa 1517 to 1531) helicase motifs, or the B "SGFPMTVIFNS" (aa 2555 to 2565) and C

"YGDD" (aa 2603 to 2606) RdRp motifs. However, polyprotein cleavage sites are much less conserved between members of the Sequivirus genus [START_REF] Sanfaçon | Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses[END_REF]), so that it was not possible to propose candidates for all tentative cleavage sites of the JG1-B polyprotein (Fig. 2B and Table 2) from a comparison with those reported for PYFV [START_REF] Turnbull-Ross | Sequence analysis of the parsnip yellow fleck virus polyprotein: evidence of affinities with picornaviruses[END_REF]. Nevertheless, four candidate sites liberating the P1 protein and the three CP subunits could be proposed (N/P, Q/G, Q/G and Q/A) at aa position 383/384, 575/576, 799/800 and 1035/1036 of the polyprotein, respectively (Fig. 2B and Table 2). With its 79.8% overall nt identity with the JG1-B sequence, the partial JG1-A isolate sequence revealed complete conservation of the genome features outlined above.

Genome organization of the LeMoV isolate identified in the JG2 lettuce

The genome ends of the 8,927 nt contig for the LeMoV isolate assembled from the sequence data generated for the JG2 lettuce were determined by 5'-RACE and 3' LD-polyT / LD-prime PCR. As for the JG1-B sequivirus, it was necessary to add a poly-A tail at the 3' genome end.

The completed JG2 LeMoV genome has been deposited in GenBank (MT348707) and is 9,927 nt long and has 5' and 3' UTRs of respectively 158 and 704 nt (Fig. 2C). It harbors a single large ORF of 9,066 nt encoding a predicted polyprotein of 3,022 aa (336.1 kDa), which has all the expected conserved protein motifs at the expected positions. Indeed, all aa sequences of motifs are similar to those identified for both isolates JG1-A and JG1-B except for the motif C of the helicase region for which a serine replaces an arginine at the 6 th position. As for the JG1-B sequence, it was only possible to predict the four tentative cleavage sites liberating the P1 protein and the three CP subunits (Fig. 2C and Table 2). Two out of four predicted cleavage sites Q/G (577/578, between CP1 and CP2) and Q/A (1037/103, between CP3 and Helicase) are identical to those predicted for JG1-B.

Genetic diversity of JG1-A/JG1-B-like isolates and of lettuce mottle virus (LeMoV)

Two nearly complete Sequivirus genome sequences related to the JG1-A/JG1-B isolates were assembled from the reads obtained from a symptomatic cultivated lettuce (21-015C, cv.

unknown) collected in southern France in 2022. These two contigs are respectively 10107 nt (21-015C-A, OQ446446) and 10066 nt (21-015C-B, OQ446447) and had an average coverage of respectively 460 and 343x (Table 1). No specific effort was made to complete the between 11.1% and 14.4% nt divergence (2.0-3.5% aa divergence) with these 5 novel near complete LeMoV sequences, which in turn show between 11.4% and 15% nucleotide divergence (2.2-3.9% aa divergence) with each other. All conserved protein motifs and predicted cleavage sites are identical for the various LeMoV isolates (Table 2).

Genomic organization of a DaYMV isolate identified in a Slovak dandelion

As described above, two viral contigs with homologies to sequiviruses could be assembled from the reads obtained from the dandelion RNA24P plant from Slovakia. These contigs respectively integrate 3,824 and 1,003 reads, corresponding to average coverages of 60-fold and 15-fold, respectively and showed the highest homology (49% nt identity) with CNDV (Table 2). Further assembly efforts yielded two large scaffolds named RNA24P-1 and RNA24P-2 of 9,786 and 9,795 nt long, respectively, and sharing 77% and 81.7 % nt sequence identity (95% and 96.2 % aa), respectively, with the only available DaYMV partial sequence (DQ675189, 788 nt long). No specific efforts were made to determine the 5' and 3' terminal regions of the nearly complete RNA24P-1 and RNA24P-2 genomes and both large contigs have been deposited in GenBank (MT559283 and MT559284, respectively). These results constitute the first identification of DaYMV isolates in dandelion in Slovakia. The RNA24P-1 genome organization is similar to that of other sequiviruses, with a single large ORF of 9,102 nt encoding a polyprotein of 3,035 aa (338.7 kDa). The incomplete 5' and 3' UTRs are respectively 149 nt and 533 nt long (Fig. 2D), indicating that the amount of missing sequence information is likely limited. The RNA24P-2 genome has an ORF of 9,105 nt encoding a polyprotein which is one aa longer than that of RNA24P-1 in the 3C-like protease domain. All 2), with an M/S polymorphism between the two isolates at the +1 position of the CP2-CP3 cleavage site.

Sequence comparisons and phylogenetic relationships of the various viruses identified in the analyzed wild and cultivated lettuces

To clarify the taxonomic position of these various viruses identified above, sequence comparisons and phylogenetic analyses were performed either with the taxonomically relevant Pro-Pol region (defined as spanning the part of the polyprotein between the conserved CG protease and GDD polymerase motifs) or with the coat protein subunits for all viruses in the genera Waikavirus and Sequivirus, as well as for representative members of the genera Cheravirus, Comovirus, Cholivirus, Fabavirus, Nepovirus, Satsumavirus, Stralaravirus, Torradovirus and for unassigned viruses in the Secoviridae family [START_REF] Sanfaçon | Proposed revision of the family Secoviridae taxonomy to create three subgenera, "Satsumavirus", "Stramovirus" and "Cholivirus", in the genus Sadwavirus[END_REF]) (Fig. 3A and3B). The trees unambiguously placed the JG1 Waikavirus with other genus members with strong bootstrap support and the JG1-A/JG1-B-like and LeMoV sequences within the Sequivirus genus, again with strong bootstrap support (Fig. 3A and3B), confirming the assignation of these various viruses to this genus. In the case of the sequiviruses, only very short partial genome sequences were previously available for LeMoV and DaYMV [START_REF] Jadao | Further characterization of two sequiviruses infecting lettuce and development of specific RT-PCR primers[END_REF]). Integration of these partial sequences in the phylogenetic trees confirm the identification of the JG2 isolate as LeMoV, together with the P22, SP, LIB3-A, LIB3-B and 21-015C isolates, with a tight, bootstrap-supported clustering (Fig. 3A) and

shows that DaYMV unambiguously clusters away from the bootstrap-supported cluster formed by the the JG1-A, JG1-B, 21-015C-A and 21-015C-B isolates, ruling out their interpretation as DaYMV isolates (Fig. 3A).

The aa sequence identity levels between the JG1 Waikavirus and other waikaviruses are below 64% and 56% for the Pro-Pol region and for the CPs, respectively (Supplementary Table S2). Considering the species molecular discrimination criteria in the Secoviridae family (less than 80% aa sequence identity in the Pro-Pol region and less than 75% identity in the CPs) [START_REF] Sanfaçon | Secoviridae: a proposed family of plant viruses within the order Picornavirales that combines the families Sequiviridae and Comoviridae, the unassigned genera Cheravirus and Sadwavirus, and the proposed genus Torradovirus[END_REF][START_REF] Thompson | ICTV Virus Taxonomy Profile: Secoviridae[END_REF], the isolate identified in the JG1 lettuce should be considered as belonging to a novel species, for which the name lettuce waikavirus 1 (LWV1) is proposed.

Concerning the JG1-A/B and 21-015C-A/B isolates, they share with each other 86.2-97.5% and 92.1-99.7% aa sequence identity in their Pro-Pol and CPs, respectively. They should thus be considered as isolates belonging to the same virus species. As compared with other known sequiviruses, they share at most 76.9% identity in the Pro-Pol region (with LeMoV isolates), which is below the 80% species demarcation criterion [START_REF] Sanfaçon | Secoviridae: a proposed family of plant viruses within the order Picornavirales that combines the families Sequiviridae and Comoviridae, the unassigned genera Cheravirus and Sadwavirus, and the proposed genus Torradovirus[END_REF][START_REF] Thompson | ICTV Virus Taxonomy Profile: Secoviridae[END_REF]) (Supplementary Table S3). However, when comparing the CPs, an aa identity value of 82.2-84.2% is observed with LeMoV isolates, higher than the 76% species cut-off value. This suggests that other species discrimination criteria need to be taken into consideration [START_REF] Thompson | ICTV Virus Taxonomy Profile: Secoviridae[END_REF], in order to be able to decide on their taxonomic situation. We therefore decided to compare the experimental host range of LeMoV with that of the JG1-A and JG1-B isolates.

Comparative host range analysis for various sequiviral isolates

Despite repeated attempts using various buffers and inoculation conditions, all efforts to transmit LWV1 from the JG1 plant to lettuce (cv. Trocadéro) failed (data not shown). The same was observed for LeCV1 [START_REF] Svanella-Dumas | Complete genome sequence of lettuce chordovirus 1 isolated from cultivated lettuce in France[END_REF]). These observations are consistent with the notion that waikaviruses are not mechanically sap-transmissible because they are confined to phloem tissue [START_REF] Reavy | Parsnip yellow fleck and rice tungro sperical viruses resemble picornaviruses and represent two genera in a proposed new plant picornavirus family (Sequiviridae)[END_REF]). On the other hand, variants JG1-A and JG1-B were readily transmitted from the JG1 plant and induced systemic mosaic and star chlorosis symptoms on inoculated lettuce (cv. Trocadéro) (Figure 1F). These initial transmission efforts also allowed to separate the JG1-A and JG1-B isolates as demonstrated using isolate-specific RT-PCR assays (Supplementary Table 1). The JG1-A and JG1-B isolates, as well as the JG2 LeMoV isolate, were sap-inoculated to plants belonging to ten species in four families, some of which are reported as differential hosts between LeMoV and DaYMV (Jadao et al. 1997[START_REF] Bos | Further characterization of dandelion yellow mosaic virus from lettuce and dandelion[END_REF]; Table 3). The expression of symptoms was monitored over a one-month period and the presence of the various viruses was verified in non-inoculated upper parts of test plants by specific RT-PCR assays. The results of these experiments are presented in Table 3.

In summary, the JG1-A and JG1-B variants were found to accumulate in six and seven of the ten tested species, respectively. For both isolates, systemic mosaic symptoms were observed on lettuce (cv. Trocadero), endive (Chicorium endivia cv. Anjou) (Fig. 1E) and common sowthistle (Sonchus olearaceus), while asymptomatic accumulation was observed in N. benthamiana, N. clevelandii and Gomphrena globosa (very occasionally only for JG1-A).

JG1-B, but not JG1-A, also accumulated in C. quinoa in which it induced yellow systemic spots (Figure 1D).

In parallel experiments, inoculation of the JG2 LeMoV isolate yielded results only partially comparable to those reported by Jadao et al. (1997) for the Brazilian AF197 isolate since contrary to AF197, JG2 caused systemic mosaic symptoms in C. endivia and only caused symptomless infection in G. globosa (Table 1). Another noteworthy difference is that contrary to AF197, which systemically infected and caused local lesions and systemic symptoms in C.

quinoa, JG2 almost never infected this host. However, a C. quinoa plant was identified with JG2 infection in independent experiments than those reported in Table 3. The virus from this plant could then readily be transmitted to C. quinoa but with only a drastically reduced efficiency to lettuce (not shown).

Taken together these results show that similar to the situation with LeMoV or with DaYMV, there is significant inter-isolate variability in the host range of the JG1-A and JG1-B isolates.

The only host that consistently separated the JG1-A and JG1-B isolates from LeMoV isolates was N. clevelandii, which was symptomlessly infected by the former, but not by the latter.

Differences also exist with DaYMV, for which multiple isolates are reported to cause local lesions in C. amaranticolor and to be unable to infect S. oleraceus (Table 3, [START_REF] Bos | Further characterization of dandelion yellow mosaic virus from lettuce and dandelion[END_REF].

DISCUSSION

In the current study, the use of HTS data in combination with 3' and 5' RACE and confirmation by RT-PCR assays for regions of low coverage allowed us to assemble complete genomic sequences for three Secoviridae family members from symptomatic lettuces cultivated in France. Similar approaches were also used to obtain near-complete genome sequences for additional isolates of the same viruses from wild or cultivated lettuces in France or in Brazil as well as the first near-complete genomes for yet another Secoviridae member, DaYMV from a dandelion from Slovakia.

For the Waikavirus found in the cultivated JG1 lettuce, its genome characterization and phylogenetic analysis show that it belongs to a new species, for which the name lettuce waikavirus 1 is proposed. The unsuccessful results at mechanical transmission suggest that like RTSV, this new virus could be confined to phloem tissues and not mechanically transmissible [START_REF] Reavy | Parsnip yellow fleck and rice tungro sperical viruses resemble picornaviruses and represent two genera in a proposed new plant picornavirus family (Sequiviridae)[END_REF]. The biology and epidemiology of waikaviruses remain poorly understood, likely in part due to their phloem restriction and reliance on specific arthropod vectors for efficient transmission [START_REF] Reddick | Nucleotide sequence and taxonomy of maize chlorotic dwarf virus within the family Sequiviridae[END_REF]. Some waikaviruses also serve as helper viruses for sequiviruses or caulimoviruses [START_REF] Elnagar | The role of the helper virus, anthriscus yellows, in the transmission of parsnip yellow fleck virus by the aphid Cavariella aegopodii[END_REF][START_REF] Thompson | ICTV Virus Taxonomy Profile: Secoviridae[END_REF]). However, some other sequiviruses such as DaYMV and LeMoV are reported to be transmitted by aphids in the absence of a helper virus [START_REF] Jadao | Further characterization of two sequiviruses infecting lettuce and development of specific RT-PCR primers[END_REF]). In the case of the JG1 plant, given the mixed infection involving both LWV1 and the JG1-A and JG1-B sequivirus isolates, it is not possible to know whether transmission complementation effects exist or not between these various Secoviridae members. However, the identification of the 21-015C-A and -B isolates in a wild lettuce and in the absence of a Waikavirus suggests that similar to LeMoV these isolates do not require the assistance of a Waikavirus for transmission.

Considering the 80% cut-off molecular criterion in the Pro-Pol region defining species within the family Secoviridae [START_REF] Sanfaçon | Secoviridae: a proposed family of plant viruses within the order Picornavirales that combines the families Sequiviridae and Comoviridae, the unassigned genera Cheravirus and Sadwavirus, and the proposed genus Torradovirus[END_REF][START_REF] Thompson | ICTV Virus Taxonomy Profile: Secoviridae[END_REF], the JG1-A, JG1-B, 21-015C-A and 21-015C-B isolates should all be considered as belonging to a single new species in the genus Sequivirus. However, when considering the 76% cut-off value that applies to the CPs, they should be considered as isolates of LeMoV. They are however unambiguously different from DaYMV, the only sequivirus known to date from lettuce in Europe. At the same time, the JG2, P22, LIB3-A, LIB3-B and 21-015C isolates from cultivated and wild lettuces are unambiguously European LeMoV isolates that tightly cluster with LeMoV sequences from South America (Figure 3A & B). The host range analysis identified a single consistent difference between LeMoV isolates and the JG1-A and JG1-B isolates, with the former being unable to infect N. clevelandii (Table 3). Considering together the phylogenetic analyses (Fig. 3), the species molecular criterion met for the Pol-Pro region, the differences in the predicted cleavage sites liberating the 3 CPs (Fig. 2 and S4.

  greenhouse and was collected in 2015 near Angers (western France). The last (21-015C, variety unknown) was collected in southern France in 2022. The mosaic symptoms on the JG2 plant, shown in Fig 1A, had more pronounced necrosis than those on the JG1 plant, while besides mosaic, the 21-015C plant showed stunting and leaf deformation. Several isolates of lettuce mottle virus (LeMoV) were analyzed. The P22 isolate was obtained from an asymptomatic wild lettuce plant (L. serriola) collected in spring 2010 as part of a plant virus metagenomic survey in Villenave d'Ornon (southwestern France) (Svanella-Dumas et al. 2018). The second isolate (SP) was from a cultivated lettuce collected in 2013 in Piedade County, Sao Paulo State, Brazil. This isolate was propagated in Chenopodium quinoa or lettuce (cv. Trocadéro) plants by sap inoculation before HTS analysis. The third LeMoV isolate, LIB3, was from a wild lettuce (Lactuca saligna) showing leaf yellowing and discolorations collected in 2020 in Villenave d'Ornon.

  . They are relatively conserved between genus members and generally involve a glutamine (Q) residue at the -1 position(Sekiguchi et al. 2005;[START_REF] Sanfaçon | Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses[END_REF]. Considering the homologies between the lettuce virus polyprotein and those of other waikaviruses, it was possible to propose tentative cleavage sites for its polyprotein using a ClustalW-generated multiple alignment of the various polyproteins (Fig.2 and Table 2). A comparison of the amino acid sequence at each of the proteolytic cleavage sites of the lettuce virus (hereafter referred to as LWV1-JG1) showed that glutamine was conserved at the -1 position, with different amino acids in the +1 position (Q/S, Q/T and Q/D) depending on the cleavage site. Comparisons between the LWV1-JG1 polyprotein sequence and those of other waikaviruses confirmed the presence of the expected conserved domains in the Hel-Pro-Pol module that is commonly used to characterize picorna-like viruses (Le[START_REF] Pavan | Virus diseases of lettuce in Brazil[END_REF]). This includes the A "GAPGVGKS" (aa 1806 to 1813), B "DD" (aa 1857-1858) and C "KGKHCTSKYVFSCTN" (aa 1893 to 1907) helicase motifs (Le[START_REF] Pavan | Virus diseases of lettuce in Brazil[END_REF], the 3C-like protease catalytic triad of histidine, aspartate and cysteine[START_REF] Reddick | Nucleotide sequence and taxonomy of maize chlorotic dwarf virus within the family Sequiviridae[END_REF] present at amino acids 2670, 2707 and 2801 and the A "DYAKFDGIGSP" (aa 3149 to 3159), B "SGFSMTVIFNS" (aa 3210 to 3220) and C

5

  ' and 3' genome UTRs. The two sequences show 23.7% nt divergence with each other and respectively 20.8% and 25% divergence with JG1-A. On the other hand, 21-015C-A shows only 8.6% divergence with JG1-B, while the corresponding value for 21-015C-B is 23.9%. Genomic organization and key protein motifs are conserved between these two new isolates Svanella-Dumas et al., 15; Phytopathology and the JG1 isolates, with only one polymorphism observed at the P1/CP1 tentative cleavage site (Table 2) Nearly complete genome sequences were obtained for five other LeMoV isolates, P22 assembled from the reads obtained from an asymptomatic wild lettuce plant (L. serriola) collected in spring 2010 as part of a plant virus metagenomic survey in Villenave d'Ornon (southwestern France) (Svanella-Dumas et al. 2018), SP assembled from the reads from a cultivated lettuce collected in 2013 in Sao Paulo State, Brazil, 21-015C assembled from the reads of the cultivated lettuce mentioned above and LIB3-A and LIB3-B assembled from the reads from a wild Lactuca saligna collected in southwest France in 2020. The P22 contig of 9,012 nt (GenBank MT348708) integrates 2,517 reads (78x average coverage), while a large part of the SP isolate genome (8,366 nt) was determined by primer walking along the genome (GenBank MT348709). The LIB3-A (OQ446443) and LIB3-B (OQ446444) sequences are 9150 nt long while the 21-015C sequence (OQ446445) is 9807 nt long but contains a short 24 nt internal gap at the position of the C rich region in which a similar gap was closed by PCR for the JG2 sequence. No specific efforts were made to determine the missing terminal sequences for any of these five LeMoV sequences. The JG2 genomic sequence shows

  polyprotein conserved amino acid motifs are identical to those identified for LeMoV isolates, except for the helicase A motif in which a lysine replaces a serine at position 2. As for LSMV and LeMoV, it was only possible to propose candidates for the first four polyprotein cleavage sites of DaYMV (K/P, Q/G, Q/M/S and Q/G at aa positions 388/389, 580/581, 804/805 and 1041/1042, respectively; Fig.2D and Table

Fig. 1 .

 1 Fig. 1. Symptoms caused by lettuce mottle virus (LeMoV) and lettuce star mosaic virus (LSMV) in various host plants. A, Necrotic mosaic symptoms observed on the original field-grown JG2 lettuce (cv. Guétary). B, Systemic necrotic lesions of LeMoV JG2 isolate on Chicorum endivia cv. Anjou 13 days post-inoculation (dpi). C, Systemic mosaic symptoms of LeMoV JG2 isolate on lettuce cv. Trocadéro at 13 dpi. The leaf on the left is from an uninoculated control plant. D, Ssymptoms of lettuce star mosaic virus (LSMV) isolate JG1-B on Chenopodium quinoa at 36 dpi. E, Symptoms of lettuce star mosaic virus (LSMV) isolate JG1-A on C. endivia cv. Anjou at 19 dpi. F, Symptoms of LSMV JG1-A (left) and JG1-B (right) in lettuce cv. Trocadéro.

Fig. 2 .

 2 Fig. 2. Schematic representation of the genomic organization drawn to scale of A, lettuce waikavirus 1 (LWV1) isolate JG1. B, lettuce star mosaic virus (LSMV) isolate JG1-B. C, lettuce mottle virus (LeMoV) isolate JG2. D, dandelion yellow mosaic virus (DaYMV) isolate RNA24P-1, partial. The short horizontal lines show the 5' and 3' untranslated regions, respectively. The large box is the long open reading frame with the coordinates of the beginning and the end mentioned. Predicted cleavage sites and their positions are shown as triangles under predicted mature proteins and uncertain cleavage sites by a question mark. P1, hypothetical protein 1; CP1, CP2, CP3, coat proteins 1, 2, and 3; NTB, NTP-binding helicase; PRO, 3C-like protease; RdRp, RNA-dependent RNA polymerase are indicated within the polyprotein. Predicted ORFX is indicated by a box for LWV1-JG1.

Fig. 3 .

 3 Fig. 3. Unrooted phylogenetic trees reconstructed using A, the Pro-Pol region, and B, the coat protein amino acid sequences from the members of the genera Waikavirus and Sequivirus, one representative species of other genera in family Secoviridae, and unassigned plant viruses showing sequence homology with the members of Secoviridae. The sequences of the viruses determined in the present work are indicated in bold font with black dots. The trees were constructed using the neighbor-joining method and the bootstrap values (500 replicates) greater than 70% are indicated next to the branches. The scale bar represents 10% aa divergence. The abbreviations and the accession numbers of the viruses used in this work are mentioned in Supplementary TABLES4.

  For JG2, the largest viral contig identified from dsRNA-derived reads, was 1,996-nt long (96x average coverage, 891 reads) and showed 50% nt identity with CNDV (data not shown).

	Given the very incomplete assembly of the viral genome, a second HTS run was performed
	on this viral isolate using total RNAs purified from infected N. benthamiana leaves. Using
	this second dataset, evidence for a single viral infection was obtained. A single long viral
	contig was obtained and further extended by rounds of reads mapping into an 8,927-nt long
	scaffold integrating 119,029 viral reads (1,790x average coverage) and sharing 50% nt
	identity with CNDV (Table 1). Interestingly, this scaffold shared 85.4 and 86.3% nt identity,
	respectively. Following the extension of these two contigs by several rounds of reads mapping
	and aggregation of additional smaller contigs, two scaffolds of respectively 8,961 nt and
	9,158 nt, were reconstructed, showing 79.8% nt identity. Taken together these results showed

and showing the highest average coverage (449x), showed the highest BlastN identity (49% nt identity) with bellflower vein chlorosis virus (BVCV, Seo et al. 2015), a recently described member of the genus Waikavirus. A second contig, integrating 12,327 reads for a 283x average coverage, showed the closest homology (54% nt identity) with carrot chordovirus 1 (Adams et al. 2014). The complete genome of this viral isolate has since been completed, allowing the description of a new viral species, lettuce chordovirus 1 (LeCV1, Svanella-Dumas et al. 2018) in the family Betaflexiviridae. The last two contigs integrating respectively 4,198 and 5,227 reads (average coverage of respectively 92x and 102x) showed highest BlastN nt identities of 54% with parsnip yellow fleck virus (PYFV, Turnbull-Ross et al. 1993) and 47% with carrot necrotic dieback virus (CNDV, Menzel et al. 2008), two members of the Sequivirus genus, that the original JG1 lettuce had a mixed infection involving LeCV1, a tentatively novel Waikavirus and two related and tentatively novel sequiviruses. respectively (98.4 and 97.9% aa identify, respectively) in a 562 nt region shared with the two partial LeMoV sequences available (DQ675190 and DQ675191), suggesting that the viral isolate identified in the JG2 plant might represent the first identification of LeMoV in cultivated lettuce outside of south America.

TABLE 1 .

 1 Table2) and the host range difference, we suggest that the JG1-A/B and 21-015C-A/B isolates could all be considered as belonging to a distinct species, for which the name lettuce star mosaic virus (LSMV) is proposed. LSMV is clearly pathogenic in lettuce, inducing star mosaic symptoms that may turn to pin-point or star necrosis and are quite similar to those caused by LeMoV, while more punctuated that the mosaics typically induced by lettuce mosaic virus (LMV). It should be noted that in the absence of fully conclusive diagnostics at the time, older reports of the presence of DaYMV in French lettuce crops[START_REF] Blancard | Maladies des salades : identifier, connaître et maîtriser[END_REF]) might, in fact, have been related to LeMoV or LSMV.Previous surveys of lettuce crops in France suggest that LSMV or LeMoV are rare, even though wild lettuce may possibly act as a reservoir in the absence of lettuce crops (hence the HTS analysis of the JG1 and JG2 plants following the observation of unusual symptoms). This situation is, at least for LeMoV, in contrast to that reported in the early 2000s in Brazil where LeMoV was considered the first or second most important lettuce virus, with a reported prevalence of 10.1%-26.1% under open field conditions in Sao Paulo state(Krause-Sakate et al. 2008;[START_REF] De Marchi | Levantamento revela a predominância do Lettuce mottle virus em três regiões produtoras de alface no Estado de São Paulo[END_REF]). However, this situation changed during the last years, withLeMoV being now more rarely found and the main viral constraint now being thrips transmitted orthotospoviruses (RKS, unpublished data). Identifying the causes underlying these differences epidemiological trends, which might be related to differences in agricultural require further investigations. The availability of complete genomic sequences for LeMoV, LSMV and DaYMV, the characterization and genome sequencing of LWV1 and the RT-PCR assays developed here should provide useful tools for such studies and for a broader and more thorough estimation of the distribution, prevalence and impact of these viruses on lettuce crops worldwide.Acknowledgments: M.G. and L.P. acknowledge support from the Scientific Grant Agency of the Ministry of Education and Slovak Academy of Sciences (VEGA 2/0030/20) and the Slovak Research and Development Agency (APVV-18-0005). The authors thank the INRAE GenoToul Platform (Toulouse, France) for high throughput sequencing, and Muriel Bahut from the ANAN technical platform of SFR QUASAV (Anger, France) for technical assistance with libraries preparations and HTS sequencing. The help of T. Mauduit and C. List of viral isolates detected in various cultivated and wild lettuces and in the RNA24P dandelion plant.For each viral contig, number of reads assigned to each isolate, the proportion of mapped reads and the average coverage are provided, together with information about the best BlastX hit. The results were obtained after HTS analysis of (a) double-stranded RNAs or (b) total RNAs from infected plants. LWV1: lettuce waikavirus 1, LeCV1: lettuce chordovirus 1, LSMV: lettuce star mosaic virus, LeMoV: lettuce mottle virus, DaYMV: dandelion yellow mosaic virus. na: does not apply TABLE 3. Infection and symptomatology of sequiviral isolates in various hosts following mechanical transmission.The number of infected over inoculated plants is indicated in parentheses. Also shown are infection and symptoms reported by[START_REF] Jadao | Further characterization of two sequiviruses infecting lettuce and development of specific RT-PCR primers[END_REF] for lettuce mottle virus (LeMoV) isolate AF197 (a) and reported in[START_REF] Bos | Further characterization of dandelion yellow mosaic virus from lettuce and dandelion[END_REF] for several isolates of dandelion yellow mosaic virus (DaYMV) (b). -, no infection; S, systemic symptoms; LL, local lesions; s, latent systemic infection; ll, latent local infection. Symptoms noted in parentheses reflect inter-isolate variability.

	Higelin (UMR NFP, INRAE Bordeaux) for taking care of plants in the greenhouse is
	gratefully acknowledged. The authors are also most grateful to the following people from
	ANSES Unité BVO (Angers, France): Michèle Visage for the first biological and molecular
	characterization of the viral isolates in the 21-015C plant, Benoit Remenant for his
	bioinformatics support and Mathieu Rolland for valuable discussions.

Amino acid positions of the cleavage sites on the polyprotein are provided in parentheses () for one representative isolate sequenced here of each species. #: a question mark indicates viruses for which it was not possible to identify a tentative dipeptide cleavage site.

Supplementary table titles and Supplementary figure captions

Supplementary


Josiane Mothe

Valentina Ciaccio

Fabio Del Frate

Davide De Santis

Mihai Ivanovici

Johan Leduc

Daniela Necşoi

Nzeh Aude

Nathalie Ndong

Marco Neptune

Recchioni

Daniela Necsoi

Aude Nzeh Ndong

Nathalie Neptune

Marco Recchioni

Zia Ullah

Giovanni Schiavon

Federica Bassini

Education and research projects for preparing young researchers for future career in Earth Observation

établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

The data availability and advances in Artificial Intelligence has led to a large increase in the market of Earth Observation (EO). It is now open to many fields and industries that look every day for new ways of operating those data. An increasing need of qualified employees is also developing, to manage at best EO data. InnEO'Space PhD programme, which aims at guiding young researchers in their future or starting career, has created modern and transferable PhD courses and learning resources to address these needs, and enhance and develop researchers' innovation-oriented skills through Earth Observation.

To contribute to PhD programs modernization, InnEO'Space created two programs, the InnEO Startech and the Machine Learning for Earth Observation InnEO Summer school. InnEO Startech aims to develop skills like innovative spirit, entrepreneurship, leadership and management, whereas the summer school focuses on open science (IPR, research management, ethics…), excellence in EO and Machine Learning, and soft and transversal skills. In support of these courses InnEO'Space PhD developed the open, integrated, and usable InnEO platform, boosting learning, professional and community resources. We developed SPOCs, Small Private Online Courses, asynchronous e-learning courses that use blended learning and short videos. Each of the SPOCs consists of 25 hours of pedagogical content in total, 1 hour for the final assessment, 8 hours are resources for students (mainly videos) and 16 hours for students' homework, intermediate tests with almost always automated correction, and individual study. Participants can acquire ECTS (European Credit Transfer Accumulation System) . A SPOC is composed of a certain number of units, each unit being semantically coherent (See Figure 1). This general structure has been adapted for each SPOC.

A SPOC has a small number of participants who have to fulfil specific requirements and whose aim is to learn specific knowledge and skills. This, and the related closer relationship with instructors and tutors, differentiates it from MOOCs (Massive Open Online Courses) [START_REF] Ruiz-Palmero | The implementation of Small Private Online Courses (SPOC) as a new approach to education[END_REF]. The small size of the group in SPOCs allows to remove the non-centrality of the students in the learning process identified as one of the main constraints in asynchronous elearning, . The blended learning course integrates collaborative and cooperative dimensions of learning, supporting individual interaction with the teacher into the learning process.

Three SPOCs have been developed and are available on the platform. One is on entrepreneurship based on our Startech program. It aims to develop skills like innovative spirit, leadership and management and relies on the Business Model Canvas. Two SPOCs are based on the lectures and practical work conceived for the summer schools: one focuses on technical skills related to Earth Observation and Machine Learning while the other is on soft and transversal skills. The importance of soft skills in the STEM (Science Technology Engineering Mathematics) field is increasingly emphasized in the era of the Industry 4.0 revolution [START_REF] Marsono | Integrating STEM (Science Technology Engineering and Mathematics) Education on Advancing Vocational Student's Creative Thinking Skills[END_REF]. We focused specifically on soft skills related to effective communication and the creation and management of a successful team. All teaching units are indexed, according to the skills, with ESCO-European Skills, Competences, Qualifications and Occupations indicator [START_REF] Commission | The ESCO Classification[END_REF].

In addition, the InnEO PRO section allows PhD students and other members such as experts in the EO and ML fields to interact with each other. It also gives access to professional community resources and job ads, proposals and research streams and promote joint research and innovation. It contains digital and soft skills selfassessment tests, as well as a tool to create an Europass CV. This tool responds to the view that it is important to give PhDs and young researchers not only training in hard and soft skills, but also tools related to knowing how to move within the working world by understanding, for example, how to handle conflicts or how to best present their own competences.

The platform and the SPOCs have been evaluated by a first series of users. After each unit, the participants answered a questionnaire about the contents and the way it is provided as well as a survey to evaluate the platform and the interaction tools. Satisfaction surveys enlighten potential areas for improvement. The development of Earth Observation, Artificial Intelligence, and Machine Learning bring a whole new range of possibilities, concerning several markets. The program we have developed aims to develop e-learning tools and processes which are transferable for any PhD program wishing to support students and young researchers in their starting career, by teaching both technical and soft skills, to improve their employability, in agreement with the current needs of the market.

The Startech modules have been reused for the European project UNIVERSEH1 , that aims to create a European university focus on space thematic. The SPOCs have been promoted by FabSpace2 , an innovation network focused on the exploitation of geo-spatial data in universities. Resources produced will be exploited by AI4AGRI, Artificial Intelligence and Earth Observation for Romania's agriculture, which aims to create a dedicated research center for AI in EO for the agricultural sector, to develop administrative and management skills for research and innovation, and to enhance the networking activities between partners through joint research, joint summer schools and workshops. The research component of the AI4AGRI 3

  project implements the principle of "learning by doing" for training the young generations of researchers in the EO domain. Addressing top priorities of EU (like agriculture, open data and open science) is an important aspect of the afore-mentioned projects.

UNIVERSEH,European Space University for Earth and Humanity Grant agreement ID; 101035795 https://universeh.eu/

Fabspace, Grant agreement ID;101006275, is an innovative network based on EO data analysis within universities https://www.irit.fr/FabSpace/

AI4AGRI, Romanian Excellence Center on Artificial Intelligence on Earth Observation Data for AGRIculture, https://ai4agri.unitbv.ro/ Grant agreement ID;101079136Figure 1 SPOC structure