Samina Mehnaz

Forman

Noreen Deeba

Baig

Salma Mukhtar

Jiaojiao Wang

Qingyue Yu

Qi Peng

Leyla Slamti

Ruibin Zhang

Shuo Hou

Didier Lereclus

Fuping Song
email: fpsong@ippcaas.cn

Deletion of the novel gene mother cell lysis X results in Cry1Ac encapsulation in the Bacillus thuringiensis HD73

Keywords: Bacillus thuringiensis, mother cell lysis, cell wall lytic enzyme C, Cry1Ac encapsulation, transcriptional regulation

The novel protein MclX (mother cell lysis X) in Bacillus thuringiensis subsp. kurstaki strain HD73 (B. thuringiensis HD73) was characterized in this work.

MclX has no known domain and its gene deletion in HD73 resulted in Cry1Ac encapsulation in the mother cell and did not influence Cry1Ac protein production or insecticidal activity. In vitro cell wall hydrolysis experiments showed that MclX cannot hydrolyze the cell wall. In mclX deletion mutants, the expression of cwlC (which encodes a key cell wall hydrolase) was significantly decreased, as shown by the β-galactosidase activity assay. MclX cannot directly bind to the cwlC promoter, based on the electrophoretic mobility shift assay (EMSA). The cwlC was reported to be regulated by σ K and GerE. However, the transcriptional activities of sigK and gerE showed no difference between HD73 and the mclX deletion mutant. It is indicated that MclX influenced cwlC expression independently of σ K or GerE, through a new pathway to regulate cwlC expression. mclX deletion could be a new approach for insecticidal protein encapsulation in Bacillus thuringiensis.

Introduction

Bacillus thuringiensis is a microbial insecticide used worldwide in agriculture [START_REF] Jouzani | Bacillus thuringiensis: a successful insecticide with new environmental features and tidings[END_REF]. The most important feature of B. thuringiensis is the formation of spores and the production of insecticidal parasporal crystals. When B. thuringiensis grown to mature, the spore and crystal protein was released from mother cell. The crystal protein is toxic to Lepidoptera, Coleoptera, Diptera and other agricultural pests Wang et al. 10.3389/fmicb.2022.951830 [START_REF] Beegle | Invitation paper (C.P. Alexander Fund): history of Bacillus thuringiensis Berliner research and development[END_REF][START_REF] Jouzani | Bacillus thuringiensis: a successful insecticide with new environmental features and tidings[END_REF]. However, the insecticidal activity of crystal protein was frequently reduced by environmental factors such as sunlight exposure [START_REF] Myasnik | Comparative sensitivity to UV-B radiation of two Bacillus thuringiensis subspecies and other Bacillus sp[END_REF][START_REF] Zogo | Impact of sunlight exposure on the residual efficacy of biolarvicides Bacillus thuringiensis israelensis and Bacillus sphaericus against the main malaria vector, Anopheles gambiae[END_REF]. Encapsulation of crystal proteins by physicochemical, mechanical, or molecular biological techniques can protect their activity from sunlight exposure [START_REF] Lereclus | Overproduction of encapsulated insecticidal crystal proteins in a Bacillus thuringiensis spo0A mutant[END_REF][START_REF] Manasherob | Protection from UV-B damage of mosquito larvicidal toxins from Bacillus thuringiensis subsp. israelensis expressed in Anabaena PCC 7120[END_REF][START_REF] Jallouli | Efficacy of olive mill wastewater for protecting Bacillus thuringiensis formulation from UV radiations[END_REF][START_REF] Chen | Novel cell wall hydrolase CwlC from Bacillus thuringiensis is essential for mother cell lysis[END_REF][START_REF] De Oliveira | Uncovering the activities, biological roles, and regulation of bacterial cell wall hydrolases and tailoring enzymes[END_REF].

Encapsulation of crystal protein by physicochemical and mechanical techniques has some advantages and disadvantages. Encapsulation by extrusion is simple to use but low productivity, encapsulation by fluidized bed is high productivity but difficult to control, encapsulation by electrospinning is easy to expand production but the media may be toxic [START_REF] De Oliveira | Uncovering the activities, biological roles, and regulation of bacterial cell wall hydrolases and tailoring enzymes[END_REF]. Crystal protein encapsulation in mother cell by molecular biological techniques was proved to be a good strategy to improve Cry protein stability under ultraviolet (UV) radiation [START_REF] Sanchis | Development and field performance of a broad-spectrum nonviable asporogenic recombinant strain of Bacillus thuringiensis with greater potency and UV resistance[END_REF][START_REF] Zhou | Screening of cry-type promoters with strong activity and application in Cry protein encapsulation in a sigK mutant[END_REF]. The mother cell wall can protect Cry protein from inactivation by UV radiation. Deletion of sigK led to blocking mother cell lysis, encapsulating crystal proteins within the mother cell wall. This technology not only constructed spore-free strain, which had no pollution to the environment and no competition with wild B. thuringiensis strains, but also improved the UV resistance of crystal proteins in the field condition [START_REF] Sanchis | Development and field performance of a broad-spectrum nonviable asporogenic recombinant strain of Bacillus thuringiensis with greater potency and UV resistance[END_REF]. Encapsulation Cry1Ba in the mother cell of sigK mutant also increasing UV radiation resistance [START_REF] Zhou | Screening of cry-type promoters with strong activity and application in Cry protein encapsulation in a sigK mutant[END_REF]. However, some cry genes are regulated by σ K , and cry gene expressions were reduced in the sigK deletion mutant [START_REF] Adams | Molecular cloning and characterization of two genes encoding sigma factors that direct transcription from a Bacillus thuringiensis crystal protein gene promoter[END_REF][START_REF] Bravo | Analysis of cryIAa expression in sigE and sigK mutants of Bacillus thuringiensis[END_REF].

Cell wall hydrolases are important in cell wall metabolism, they are classified as glycosidases and peptidases. Glycosidases (glucosaminidase, muramidase, and lytic transglycosylase) and peptidases (amidase, endopeptidase, and carboxypeptidase) cleave different sites of cell wall peptidoglycan (Do et al., 2020a). In B. subtilis, cell wall hydrolases involved in mother cell lysis (CwlB, CwlH, CwlC) are amidases with functional redundancy. Single amidase gene deletion did not affect mother cell lysis, while cwlB cwlH cwlC deletion mutant significant reduced mother cell lysis [START_REF] Nugroho | Characterization of a new Sigma-K-dependent peptidoglycan hydrolase gene that plays a role in Bacillus subtilis mother cell lysis[END_REF]. In B. thuringiensis, CwlB and CwlC show low sequence identities with B. subtilis hydrolases. Deletion of the cwlB gene can delay lysis of mother cells [START_REF] Yang | Transcriptional regulation and characteristics of a novel N-acetylmuramoyl-L-alanine amidase gene involved in Bacillus thuringiensis mother cell lysis[END_REF]. Deletion of the cwlC can block the lysis of mother cells without influence on Cry1Ac production [START_REF] Chen | Novel cell wall hydrolase CwlC from Bacillus thuringiensis is essential for mother cell lysis[END_REF]. As the essential hydrolase in mother cell lysis, CwlC was reported to be regulated by σ K and GerE in B. thuringiensis [START_REF] Chen | Novel cell wall hydrolase CwlC from Bacillus thuringiensis is essential for mother cell lysis[END_REF]. No other genes have been reported to be deleted for encapsulation crystal protein except sigK and cwlC.

In this work, we identified a hypothetical gene whose deletion resulted in Cry1Ac encapsulation in the mother cell. The mother cell did not lysis because deletion of this hypothetical gene reduced the cwlC expression. This discovery of the regulatory mechanism of cwlC is independent from the regulatory pathway of σ K or GerE. Studies on this new gene may contribute to a deeply understanding of cwlC expression. The deletion of this new gene encapsulated Cry1Ac in the mother cell with no effect on sporulation frequency, Cry1Ac protein production, or insecticidal activity. This could be a potential new approach for Cry protein encapsulation by molecular biology techniques.

Materials and methods

Bacterial strains, growth conditions, and plasmids

Tables showed the strains (Supplementary Table 1), primers (Supplementary Table 2), and plasmids (Supplementary Table 3) used in this work. The B. thuringiensis subsp. kurstaki strain HD73 (GenBank Accession Number: NC_020238.1) was used for transformations [START_REF] Du | Bacillus thuringiensis HD73 spores have surface localized Cry1Ac toxin: physiological and pathogenic consequences[END_REF][START_REF] Liu | Complete genome sequence of Bacillus thuringiensis subsp. kurstaki strain HD73[END_REF]. All B. thuringiensis strains were cultured in SSM (Schaeffer's sporulation medium) [START_REF] Schaeffer | Catabolic repression of bacterial sporulation[END_REF] or LB (Luria-Bertani) medium under 30 • C, with 100 µg/ml Kan (Kanamycin) or 5 µg/ml Ery (Erythromycin) if needed. The E. coli strain TG1 was used for constructing the vector [START_REF] Hoffmann | Simple technique for simultaneous on-line estimation of biomass and acetate from base consumption and conductivity measurements in high-cell density cultures of Escherichia coli[END_REF]. The E. coli ET12567 was used for extracting non-methylated vectors on a large-scale and transforming into B. thuringiensis. All E. coli strains were grown in LB medium at 37 • C, with 100 µg/ml Amp (Ampicillin) if needed.

DNA manipulation and transformation

The EasyPure Plasmid MiniPrep Kit (Transgen, Beijing, China) was used to extract vectors from E. coli cells listed in Supplementary Table 3. The PrimeSTAR Max DNA Polymerase (Takara, Beijing, China) was used to perform PCR, using a Mastercycler X50 (Eppendorf). Primers were synthesized in a company (Sangon, China). The EasyPure Quick Gel Extraction Kit (Transgen, Beijing, China) was used to purified DNA fragments, the Seamless Assembly Cloning Kit (Clonesmarter, United States) was used to connect DNA fragment and the linearized vector. Plasmid was introduced into the strain HD73 by electroporation (GenePulser Xcell, Bio-Rad).

Construction of the mother cell lysis X deletion mutant strain HD ( mclX)

To delete the mclX gene in the HD73 genome, pMAD [START_REF] Arnaud | New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria[END_REF]) (a temperature-sensitive suicide plasmid) with the mclX mutation box was constructed. The mclX mutation box was amplified as follows. A 725-bp fragment upstream of mclX (mclX fragment A) which contains a 12bp overlap with the mclX 5 end, was amplified by PCR, and mclX-1 and mclX-2 were used as primers. A 677-bp fragment downstream of mclX (mclX fragment B) which starts 4-bp away from mclX 3 end, was amplified by PCR, and mclX-5 and mclX-6 were used as primers. A 1,473-bp fragment of the kan resistance gene was amplified by PCR, and mclX-3 and mclX-4 were used as primers. The overlapping PCR was used in amplifying the mclX mutation box (mclX fragment A, kan fragment, and mclX fragment B), using mclX-1 and mclX-6 as primers, and the resulting fragment of the mclX mutation box was inserted into the pMAD plasmid. Then, the recombinant plasmid was introduced into HD73. The mclX mutation box allelic replacement was performed as reported [START_REF] Yang | Transcriptional regulation and characteristics of a novel N-acetylmuramoyl-L-alanine amidase gene involved in Bacillus thuringiensis mother cell lysis[END_REF]. The mclX ORF (12-663) was replaced by kan fragment which detected by PCR with primers mclX-1 and mclX-6.

Construction of the complemented strain

To construct the complemented strain HD ( mclX:mclX), the mclX promoter and open reading frame (PmclX-mclX, 998bp) were amplified by PCR, using HFmclX-F and HFmclX-R as primers. The PmclX-mclX fragment was inserted into the pHT315 plasmid [START_REF] Arantes | Construction of cloning vectors for Bacillus thuringiensis[END_REF] to generate pHTHFmclX. The plasmid pHTHFmclX was introduced into HD ( mclX) to generate complemented strain HD ( mclX:mclX).

Construction of a PmclX-lacZ fusion strain

To analyze the transcriptional activity of the mclX promoter, a 335-bp fragment upstream of the mclX ATG start codon was amplified by PCR, using PmclX-F and PmclX-R as primers. The PmclX fragment was inserted into the pHT304-18Z plasmid [START_REF] Hervé | Structural and functional analysis of the promoter region involved in full expression of the cryIIIA toxin gene of Bacillus thuringiensis[END_REF] carrying the lacZ gene. The recombinant plasmid pHTPmclX was introduced into the HD73 strain, mutant strain HD ( sigK) or HD ( gerE).

Construction of a mother cell lysis X-His fusion protein

To express the MclX protein in an E. coli strain, an mclX gene fragment without a termination codon was amplified by PCR, using MclX-F and MclX-R as primers. The resulting fragment was inserted into the pET21b plasmid to generate pETmclX with a C-terminal His tag. The plasmid pETmclX was transformed into the BL21 strain for expressing the MclX-His protein.

Expression and purification of mother cell lysis X-His protein

The method of protein expression and purification was previously described [START_REF] Zhang | The stationary phase regulator CpcR activates cry gene expression in non-sporulating cells of Bacillus thuringiensis[END_REF]. A BL21(pETmclX) strain was grown in LB liquid medium until reaching an OD 600 of 0.7. Then, 1 mM isopropyl-beta-D-thiogalactoside (Solarbio, Beijing, China) was added to induce MclX-His expression, and bacteria were harvested after overnight growth at 18 • C and sonicated (CP750, Cole-Parmer). The supernatant was loaded onto a 2-ml suspension of Ni Sepharose 6 Fast Flow (GE Healthcare, Sweden) with wash buffer (25 mM Tris-HCl, 50 mM imidazole, 0.5 M NaCl, pH 8.0) and elution buffer (25 mM Tris-HCl, 250 mM imidazole, 0.5 M NaCl, pH 8.0). The purified MclX-His protein was analyzed by 10% SDS-PAGE (Mini protein III, Bio-rad).

β-galactosidase assays

The method of the β-galactosidase activity assay was previously described [START_REF] Miller | Experiments in Molecular Genetics[END_REF]. The error bars represent three independent experiment results.

Sporulation frequency analysis

Liquid samples (50-ml) of HD73 and HD ( mclX) were harvested at T24 (24 h after the exponential phase ended). Heated samples (under 65 • C for 20 min) and unheated samples were diluted in a gradient, coated on the LB agar plates. Then the number of grown colonies in heated and unheated sample plates was counted. The sporulation frequency is the clone number ratio of the heated sample to the unheated sample. The error bars represent three independent experiment results.

Cry1Ac protein production

The method was performed as previous described [START_REF] Zhang | A strong promoter of a non-cry gene directs expression of the cry1Ac gene in Bacillus thuringiensis[END_REF]. The wild-type strain HD73 and the strain HD ( mclX) were harvested at T24 and lyophilized into powder, same quality of samples was taken and determined by 10% SDS-PAGE (Bio-rad). The results of three independent experiments are consistent.

Cell wall preparation and hydrolysis

For cell wall preparation and hydrolysis, method was previously described [START_REF] Yang | Transcriptional regulation and characteristics of a novel N-acetylmuramoyl-L-alanine amidase gene involved in Bacillus thuringiensis mother cell lysis[END_REF][START_REF] Chen | Novel cell wall hydrolase CwlC from Bacillus thuringiensis is essential for mother cell lysis[END_REF]. CwlC added to cell wall was used as a positive control. The cell wall itself was used as a negative control, and the weight of MclX was the same as that of CwlC. The results of three independent experiments are consistent.

Bioassay of insecticidal activity

A bioassay was carried out as previous described [START_REF] Chen | Novel cell wall hydrolase CwlC from Bacillus thuringiensis is essential for mother cell lysis[END_REF]. Each test was conducted with three independent cultures replicated.

Western blot analysis

Cell lysates (the equivalent of 2 ml at the time point) were suspended in 600 µl of Tris buffer (pH 8.0) and analyzed by 10% SDS-PAGE (Bio-rad). Anti-CwlC was generated by a company (Beijing Protein Innovation Inc., China), and the second antibody (HRP-conjugated goat anti-mouse IgG) was bought (CWBiotech, Beijing, China). The results of three independent experiments are consistent.

Electrophoretic mobility shift assays

EMSA (Electrophoretic mobility shift assay) was carried out as previous described [START_REF] Shen | Transition phase regulator AbrB positively regulates the sip1Ab1 gene expression in Bacillus thuringiensis[END_REF]. The cwlC promoter was labeled with 6-carboxyfluorescein (FAM) (Supplementary Table 3). The results of three independent experiments are consistent.

Results

Deletion of the hypothetical gene HD73_RS12920 can encapsulate Cry1Ac in mother cell

To search for key genes involved in mother cell lysis, T7 transcriptome data (Tn stands for n hours after the exponential phase ended) of the strain HD73 was analyzed [START_REF] Peng | Identification of metabolism pathways directly regulated by sigma (54) factor in Bacillus thuringiensis[END_REF]. T7 stands for the late sporulation stage, at which the exosporium genes of the spore coat begin to be transcribed [START_REF] Peng | The regulation of exosporium-related genes in Bacillus thuringiensis[END_REF]. Transcription of cwlC had not started at T7, which means that mother cell lysis also had not started. Highly expressed hypothetical genes were screened (Supplementary Table 4) and genetic deletion mutants were constructed.

Fortunately, deletion of a hypothetical gene showed a phenotype in which the mother cell did not lyse. This hypothetical gene is HD73_RS12920 (which encodes a protein in NCBI with the reference sequence WP_000101499.1). HD73_RS12920 is 663-bp in length and has no known domains. According to the deletion mutant phenotype, which displayed no mother cell lysis, HD73_RS12920 was named mclX (mother cell lysis X).

In the mclX deletion mutant HD ( mclX) (method described in "Materials and Methods"), the mother cell did not lyse, while the mother cells of wild-type strain HD73 lysed at 1, 3, and 5 days after the bacterial growth reached the end of the exponential phase (Figure 1A). There was no difference in vegetative growth between the wild-type strain HD73 and HD ( mclX) (Supplementary Figure 1). To further demonstrate the role of MclX in mother cell lysis, we constructed a genetically complemented strain HD ( mclX:mclX), as described in section "Materials and methods." In the mother cell lysis stage, most wild-type and HD ( mclX:mclX) mother cells have lysed (Supplementary Figure 2). The results showed that MclX played an important role in mother cell lysis.

There were no differences between the wild-type strain HD73 and HD ( mclX) in sporulation frequency or Cry1Ac protein production at T24 (Figures 1B,C). The second instar larva of Plutella xylostella was used to determine the insecticidal activity of the wild-type strain HD73 and HD ( mclX). P. xylostella larvae were fed prepared cabbages treated with a mixture of spores and crystals of the wild-type strain or the HD ( mclX) strain. The LC 50 of the wild-type strain HD73 was 8.89 µg/ml and the LC 50 of the HD ( mclX) strain was 10.2 µg/ml, displaying no significant differences (Table 1). Therefore, the deletion of the mclX encapsulated Cry1Ac in HD73 mother cells, without effect on sporulation frequency, Cry1Ac protein production, or insecticidal activity. CwlC protein (known cell wall hydrolase) plus cell wall was used as a positive control (squares), and the cell wall itself was used as a negative control (circles).

Transcriptional regulation of the mother cell lysis X gene

To test whether MclX could be a cell wall hydrolase, the MclX protein hydrolyzed cell wall was analyzed in vitro. The MclX-His protein was expressed and analyzed by SDS-PAGE, its molecular weight was approximately 30 kDa (Figure 3A). The MclX-His protein and the wide-type strain HD73 cell wall were incubated together. The optical density at 540 nm (OD 540 ) showed cell wall turbidity. CwlC is a known hydrolase that can reduce the cell wall turbidity (Figure 3B, squares). However, there was no reduction in the OD 540 values of MclX Mother cell lysis X gene deletion decreases cwlC expression.

Previous research indicated that cwlC is controlled by σ K and positively regulated by GerE [START_REF] Chen | Novel cell wall hydrolase CwlC from Bacillus thuringiensis is essential for mother cell lysis[END_REF]. Since HD ( mclX) exhibited a similar phenotype to HD ( cwlC), whether the deletion of the mclX affected the transcriptional activity or protein expression of CwlC was analyzed.

The transcriptional activity of cwlC was investigated in HD ( mclX). β-galactosidase activity assays indicated that cwlC expression significantly decreased in HD ( mclX), compared to that in wild-type strain (Figure 4A). Whether MclX could directly bind to the cwlC promoter was tested by EMSA. MclX was found to be unable to directly bind to the cwlC promoter Wang et al. 10.3389/fmicb.2022.951830 (Figure 4B). It is speculated that mclX deletion might affect cwlC transcription levels by affecting transcriptional activities or protein functions of σ K and GerE. To investigate this, the transcriptional activities of sigK and gerE in HD ( mclX) and the wild-type strain were analyzed, showing no differences (Figures 4C,D). The protein functions of σ K and GerE in HD ( mclX) were detected. bxpB, which was found to be regulated by σ K and GerE [START_REF] Peng | The regulation of exosporium-related genes in Bacillus thuringiensis[END_REF], showed no differences in transcription between the wild-type strain and HD ( mclX) (Supplementary Figure 5). Thus, MclX indirectly affected cwlC transcriptional activity, not by affecting σ K or GerE.

Moreover, CwlC protein production was investigated in HD ( mclX). In HD ( mclX) cell lysate, CwlC protein was barely visible in the anti-CwlC immunoblot, whereas significant amounts of CwlC protein were produced in the wild-type strain cell lysate (Figure 5), with the same bacterial volumes taken at the same time point and using the same loading volumes.

Altogether, CwlC expression was dramatically decreased and barely detectable in HD ( mclX).

Discussion

In this work, the novel gene mclX was characterized, whose deletion resulted in Cry1Ac encapsulation in the mother cell of the HD73 strain (Figure 1A). MclX was unable to hydrolyze the cell wall in vitro (Figure 3B), and it is a newly identified mother cell lysis-associated protein which is not an amidase. MclX can be found not only in the B. cereus group but also in some S. pneumoniae strains (Supplementary Figure 4). S. pneumoniae is a Gram-positive human pathogen leading to global health problems [START_REF] Engholm | A visual review of the human pathogen Streptococcus pneumoniae[END_REF]. Its MclX homologous protein shares 100% similarity with that of HD73, and the role of MclX in S. pneumoniae cell lysis maybe very interesting and warrants further studies.

cwlC encodes a key cell wall hydrolase, and its deletion resulted in Cry1Ac encapsulation in the HD73 strain. Only a few reports about the regulation of cwlC, while the regulation mechanisms of some other hydrolases have been reported (Do et al., 2020a). Transcription of the hydrolases lytE and cwlO is regulated by the WalKR signal transduction pathway in B. subtilis [START_REF] Dobihal | Homeostatic control of cell wall hydrolysis by the WalRK two-component signaling pathway in Bacillus subtilis[END_REF]. The endopeptidase EagA is regulated through bacterial second messenger molecule c-di-GMP signaling in Erwinia amylovora [START_REF] Kharadi | Cyclic-di-GMP regulates autoaggregation through the putative peptidoglycan hydrolase, EagA, and regulates transcription of the znuABC zinc uptake gene cluster in Erwinia amylovora[END_REF]. Protein interactions with hydrolases can activate [START_REF] Uehara | LytM-domain factors are required for daughter cell separation and rapid ampicillin-induced lysis in Escherichia coli[END_REF][START_REF] Uehara | Daughter cell separation is controlled by cytokinetic ring-activated cell wall hydrolysis[END_REF][START_REF] Yang | An ATP-binding cassette transporter-like complex governs cell-wall hydrolysis at the bacterial cytokinetic ring[END_REF][START_REF] Domínguez-Cuevas | Differentiated roles for MreB-actin isologues and autolytic enzymes in Bacillus subtilis morphogenesis[END_REF][START_REF] Meisner | FtsEX is required for CwlO peptidoglycan hydrolase activity during cell wall elongation in Bacillus subtilis[END_REF]Do et al., 2020b) or inhibit hydrolase activities [START_REF] Clarke | The vertebrate lysozyme inhibitor Ivy functions to inhibit the activity of lytic transglycosylase[END_REF]. The mother cell of the HD ( mclX) strain did not lyse because the cwlC expression decreased (Figure 4A). The expression of mclX was detected earlier than that of cwlC (Figures 2B,4A The HD ( mclX) strain displayed no mother cell lysis and did not have an altered sporulation frequency, Cry1Ac protein production, or insecticidal activity (Figure 1 and Table 1). mclX deletion could be a new biotechnological approach in the encapsulation of Cry proteins resistant to adverse environmental factors. The deletion of cwlC or sigK in the Bacillus thuringiensis var. israelensis (Bti) strain did not block mother cell lysis [START_REF] Xu | Characteristics of the sigK deletion mutant from Bacillus thuringiensis var. israelensis strain Bt-59[END_REF][START_REF] Huang | Characterization of CwlC, an autolysin, and its role in mother cell lysis of Bacillus thuringiensis subsp. israelensis[END_REF], because other cell wall hydrolases, like CwlE, are involved in mother cell lysis in Bti [START_REF] Huang | Characterization of CwlC, an autolysin, and its role in mother cell lysis of Bacillus thuringiensis subsp. israelensis[END_REF]. However, the deletion of the mclX homolog gene in Bti also did not block mother cell lysis (Supplementary Figure 6). In comparison to the deletion of cwlC in Bti, the deletion of the mclX homolog gene in Bti led to more effective delays in mother cell lysis (Supplementary Figure 6). Thus, insecticidal protein encapsulation in mother cells requires targeted strategies in different B. thuringiensis strains.

Conclusion

Deletion of mclX provides a novel approach for encapsulation of Cry protein in B. thuringiensis. MclX has no known domains and it is a key protein in the mother cell lysis of B. thuringiensis strain HD73. It is a discovery of blocking mother cell lysis which not cause by cell wall hydrolases. This strategy could be utilized to genetically modify the B. thuringiensis products for enhancing UV resistance. Wang et al. 10.3389/fmicb.2022.951830 research. All authors contributed to the article and approved the submitted version.

FIGURE 1

 1 FIGURE 1 Phenotype analysis of the HD ( mclX) strain. (A) Phenotype of the wild-type strain HD73 and HD ( mclX) at 1, 3, and 5 days after the bacterial growth reached the end of the exponential phase. Scale bars, 5 µm; (B) sporulation frequency of the wild-type strain HD73 and HD ( mclX). Error bars show the standard error of the mean; (C) Cry1Ac protein production in the wild-type strain HD73 and HD ( mclX). Cry1Ac production bands are marked by arrows. Lane M, protein marker (26616).

  MclX has no known domain and a molecular size of 25.6 kDa. Secondary structure alignment (PSIPRED) 1 revealed the presence of several α-helixes and β-strands in MclX (Supplementary Figure3). In the B. thuringiensis HD73 genome, mclX has an upstream gene, RS12915, which encodes a YozE family protein, and a downstream gene, RS12925, which encodes a hypothetical protein (Figure2A). In the B. cereus group, MclX is very conserved (Supplementary Figure 4). Streptococcus pneumoniae, Bacillus subtilis, and Rhodococcus qingshengii, have a MclX homologous protein with 100, 62, and 65% similarity, respectively. The promoter region of the mclX gene (335-bp upstream of the mclX ATG start codon) was fused to the lacZ gene, and transformed into HD ( sigK), HD ( gerE), and wild-type HD73. In HD73, the transcription of PmclX initiated at T6 and reached a peak at T12 (Figure 2B, circles). In HD ( sigK), the transcriptional activity of PmclX was completely abolished (Figure 2B, triangles). In HD ( gerE), PmclX transcriptional activity was increased compared to that in wild-type strain HD73 (Figure 2B, squares). All above results demonstrate that mclX was highly expressed in the late sporulation stage, controlled by σ K , and negatively regulated by GerE. Mother cell lysis X cannot hydrolyze the cell wall in vitro HD ( mclX) displayed no mother cell lysis. MclX has no known domains, including DNA binding domains or any functional domains. Regarding its role in mother cell lysis, MclX may be an essential cell wall hydrolase or it may affect the function of the cell wall hydrolase CwlC.

1

  http://bioinf.cs.ucl.ac.uk/psipred/

FIGURE 2 mclX

 2 FIGURE 2 mclX transcription activity. (A) Gene map of the RS12915-RS12925; (B) mclX transcription activities in the wild-type strain HD73, HD ( gerE), and HD ( sigK) (HD73-PmclX, circles; HD gerE-PmclX, squares; HD sigK-PmclX, triangles). Tn means n hours after the exponential phase ended. Error bars show standard error of the mean.

FIGURE 3

 3 FIGURE 3 Characterization of the MclX protein. (A) MclX protein expressed in the recombinant strain E. coli BL21 (pETMclX) by SDS-PAGE analysis. M, protein marker, 26614; (B) purified MclX protein was unable to hydrolyze the wild-type strain HD73 cell wall. The strain HD73 cell wall was mixed with MclX protein (triangles).CwlC protein (known cell wall hydrolase) plus cell wall was used as a positive control (squares), and the cell wall itself was used as a negative control (circles).

FIGURE 4

 4 FIGURE 4 MclX indirectly affects the transcription of cwlC. (A) cwlC promoter activities in the wild-type strain HD73 and HD ( mclX) (HD73-PcwlC, circles; HD mclX-PcwlC, squares); (B) the cwlC promoter was unable to interact with the MclX protein based on EMSA. Lane 1, 1 ng FAM-labeled PcwlC probe; lanes 2-5, 1 ng labeled probe with an increasing mass of purified MclX protein; (C) sigK promoter activities in the wild-type strain HD73 and HD ( mclX) (HD73-PsigK, circles; HD mclX-PsigK, squares); (D) gerE promoter activities in the wild-type strain HD73 and HD ( mclX) (HD73-PgerE, circles; HD mclX-PgerE, squares). Error bars show standard error of the mean.

FIGURE 5

 5 FIGURE 5CwlC was determined by Western blot in wild-type HD73 and HD ( mclX) lysates. The same volumes were taken at the time points, and the same loading volumes were analyzed. CwlC-His served as positive control.

  ), although MclX cannot directly bind the cwlC promoter (Figure 4B). The way in which MclX affects the transcriptional activity of cwlC remains unknown. MclX may influence cwlC transcription by affecting other factors, and not by affecting σ K or GerE (Figures 4C,D). The function of MclX requires further research, to gain a better understanding of the regulatory mechanism of cwlC.

TABLE 1

 1 Insecticidal activities of B. thuringiensis strains against Plutella xylostella.

	Strain	LC 50 (µ g of protein/ml) 95% confidence interval
	HD73	8.89	6.95-13.96
	HD( mclX)	10.2	7.81-16.01

Frontiers in Microbiology

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Funding

This work was supported by grants from the National Natural Science Foundation (31530095 and 31772243).

Data Availability statement

The original contributions presented in this study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

Author contributions

JW and QY conducted the experiments, analyzed the data, and produced the data displays. FS designed the research. QY wrote the manuscript. LS, DL, and QP edited the manuscript. RZ, SH, and QP proposed amendments. FS and QP funded the

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2022.951830/full#supplementary-material


Adel Abbas

Michele Linardi

Etienne Vareille

Vassillis Christophides

Claudia Paris

Towards Explainable AI4EO: An Explainable Deep Learning Approach for Crop Type Mapping using Satellite Images Time Series

Keywords: Transformers, AI4EO, Dynamic Time Warping (DTW), Crop Type Mapping, Sentinel-2 data

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

INTRODUCTION

Large-scale crop type mapping is crucial for agricultural planning and management. Recently, Deep Learning (DL) models based on transformer encoders have proven their effectiveness in classifying long and dense Satellite Image Time Series (SITS) to generate accurate crop type maps [START_REF] Nyborg | Generalized classification of satellite image time series with thermal positional encoding[END_REF]. Although these models have outperformed shallow machine learning techniques, they pose challenges in terms of explainability and confidence in the results, as their input-output mapping function is usually estimated without revealing any information about the internal structure of the model. For these reasons, recently, efforts have been made to produce Explainable Artificial Intelligence (xAI) approaches able to provide additional information to end users, thus improving their understanding of the DL model results [START_REF] Gevaert | Explainable ai for earth observation: A review including societal and regulatory perspectives[END_REF]. Although xAI methods shed light on how DL models work, very little has been done to develop xAI approaches tailored to SITS [START_REF] Obadic | Exploring self-attention for crop-type classification explainability[END_REF] and to understand why these models are unable to handle domain shifting. Indeed, DL models can be highly effective in capturing and processing temporal patterns for the training region (source domain) while they typically generalize poorly to regions never seen by the network (target domain) [START_REF] Nyborg | Generalized classification of satellite image time series with thermal positional encoding[END_REF]. This is mainly due to different climate conditions and management decisions, which determines different timing of phenological phases for the same crop type in different regions [START_REF] Wang | Phenology alignment network: A novel framework for cross-regional time series crop classification[END_REF]. Several approaches have recently been proposed to focus explicitly on the temporal dimensions of SITS acquired in different areas for large-scale crop type mapping [START_REF] Wang | Phenology alignment network: A novel framework for cross-regional time series crop classification[END_REF][START_REF] Nyborg | Timematch: Unsupervised cross-region adaptation by temporal shift estimation[END_REF]. However, they focus on the data distribution shift, by completely neglecting the role played by the considered DL model. This paper presents a novel xAI approach that aims to improve the generalization capability of the existing objectbased crop classifier based on Pixel Set Encoder (PSE) and Temporal Attention Encoder (TAE) widely used to perform crop type mapping [START_REF] Nyborg | Timematch: Unsupervised cross-region adaptation by temporal shift estimation[END_REF]. Leveraging the interpretability of the model's inner workings, the proposed method automatically estimates the temporal shift of the SITS acquired in the source and the target domains by comparing their Positional Encoding (PE) outputs. To mitigate the detected temporal shift, the Dynamic Time Warping (DTW) technique is used to re-align the target PE output to the representation that the model natively adopts to discriminate crop types. By adopting this correction, the proposed approach outperforms the classification accuracy obtained by the standard network architecture without requiring any labeled data in the target domain.

PRELIMINARIES AND FORMULATION

This section summarizes the problem formulation while recalling the fundamentals of the considered DL architecture. Let TS S = {X S t } b 1 be the SITS acquired in the source domain (i.e, where the labeled data available are used to train the DL model), made up of b images, where t ∈ [1, b] represents a generic time image acquisition. Let TS T = {X T t } u 1 be the SITS acquired in the target domain (where no labeled data are available), made up of u images. In a realistic scenario, it is reasonable to assume that u ̸ = b since SITS acquired over different regions are typically characterized by unequal lengths and different temporal sampling rates [START_REF] Weikmann | Timesen2crop: A million labeled samples dataset of sentinel 2 image time series for crop-type classification[END_REF].

To generate the crop type maps in the source and target domains, we considered the object-based crop classifier PSE + TAE [START_REF] Nyborg | Timematch: Unsupervised cross-region adaptation by temporal shift estimation[END_REF]. This network has been widely used for crop-type mapping due to its ability to process the spatial, temporal and spectral information provided by SITS. The standard network architecture consists of three different encoders: (1) PSE that extracts descriptors from the spectral distribution of each input observation, (2) PE that captures the periodic relationships between elements in the input sequence, and (3) TAE that handles the temporal information of the SITS. The TAE takes as input the combination of the PSE and PE outputs and computes the attention scores (a h ) for each head h (independent input slice) as the similarity (dot product) between all keys k (t) h (for all time instant t) and the query q h , re-scaled by a softmax layer. The k h is learned by a fully connected layer, whereas the query is chosen to be a model hyperparameter. As suggested by Nyborg et al. [START_REF] Nyborg | Timematch: Unsupervised cross-region adaptation by temporal shift estimation[END_REF], we use a learnable PE that considers vectorized positions of sine-cosine encoding z(t) as input of a Gated Recurrent Units (GRU) layer that captures sequential dependencies of crop temporal evolution, providing better classification performance.

PROPOSED AI4EO APPROACH FOR LARGE SCALE CROP TYPE MAPPING

P t = DT W (A, B) (1)
When computing DTW between the two multi-dimensional PE outputs, namely pe S and pe T , we still consider a unique warping P , which draws values from a matrix M ′ , where

M ′ i,j = [ b m=1
dist(pe S m,i , pe T m,j )] and dist(pe S m,i , pe T m,j ) is the squared Euclidean distance between the pe S , pe T on the dimension m, at indexes i and j respectively [START_REF] Shokoohi-Yekta | Generalizing DTW to the multi-dimensional case requires an adaptive approach[END_REF]. In the considered implementation of the method, to represent the PE learned in the source, we compute the DTW Barycenter [START_REF] Franc ¸ois Petitjean | A global averaging method for dynamic time warping, with applications to clustering[END_REF] of the SITS acquired in the source domain. At the inference stage, when the target SITS is classified by the trained DL model, we compute the DTW distance between the PE of the instance to classify and the source Barycenter. The resulting warping path is then considered to correct the positional encoding, i.e., pe T . To that extent, we replace the values of pe T with those of pe S along the indexes contained in the warping path in order to maximize the similarity of pe T and pe S . Figure 2 shows a qualitative example of PE outputs generated for the same crop type when ingesting TS T (Figure 2a) and TS S (Figure 2d). The temporal evolutions of the Normalized Difference Vegetation Index (NDVI) computed on TS T and TS S in depicted in Figure 2b and Figure 2e, respectively. Although both samples belong to the same crop type (i.e., winter wheat), the pattern shift visible in pe T and pe S (highlighted with the vertical bars) explains and confirms the temporal shifts observed on the two NDVIs. By correcting this misalignment, it is possible to increase the accuracy in the target domain.

DATASET DESCRIPTION AND RESULTS

To test the performance of the proposed approach, we considered the public TimeMatch benchmark dataset [START_REF] Nyborg | Timematch: Unsupervised cross-region adaptation by temporal shift estimation[END_REF], which has been released for evaluating cross-region crop type mapping models. For the sake of reproducibility, the source code of our solution and other relevant material are available online 1 . The SITS, acquired in 2017 over 4 regions in Europe, are made up of Sentinel-2 images having cloud cover lower than 80%. This leads to an unequal temporal sampling and variations between the SITS acquisition time. Table 1 shows Table 1. Number of available labeled samples. While the labeled data of Denmark (DK), Austria (AT) and southern France (FR1) were used to train the models, the data in midwest France (FR2) were used for performance evaluation.

Training the number of labeled samples per crop type available per country. In the considered experimental setup, we used the labeled data available in Austria (tile 33UVP), Denmark (tile 32VNH) and southern France (tile 31TJC) to train the DL models, while the labeled data available in mid-west France (tile 30TXT) was used only for performance evaluation.

Table 2 shows the F1 score (F1%) and the Overall Accuracy (OA%) obtained on the test set by applying the Standard and the Proposed PSE+TAE architecture when using the training data from: (i) Denmark (DK), (ii) Austria (AT), (iii) southern France (FR1), and (iv) the three countries (AT+DK+FR1). As expected the best results are achieved This is due to the fact that it was able to better handle the most critical classes such as "Winter barley" and "Winter triticale" (increasing their F1% of 26% and 23%) while achieving similar accuracy on the other crop types except for "Spring barley" (the F1% decreases of 20%). Moreover, the Proposed PSE+TAE is able to improve the classification results obtained also when considering a small training set located in only one country. Compared to the standard PSE+TAE architecture, the proposed method slightly increases the OA% when training the model with the French training data, while decreasing the macro average F1% and weighted F1% of 10% and 3%, respectively. However, when training the model with the Danish and the Austrian training data, it significantly increases the OA%, macro average F1% and weighted F1% of 8% and 2%, 5% and 4%, and 12% and 2%, respectively.

CONCLUSION

This paper presented a new xAI approach designed for largescale crop mapping. The method, based on the PSE+TAE architecture, aims to improve its generalization capability by automatically comparing and re-aligning the PE outputs computed in the source and target domains without the need for any target labeled data. Preliminary results demonstrate the proposed approach outperformed the standard architecture for almost all experiments. As future developments, we aim to further study the effectiveness of the proposed approach in different regions. Moreover, we intend to further investigate the best strategy to mitigate the temporal shift detected between the PE outputs computed in the source and target domains.

Fig. 1 .

 1 Fig. 1. Schematic representation of the proposed xAI approach based on the PSE+TAE model. The method is based on 3 main components: (a) SITS Encoding, (b) Positional Encoding Correction, and (c) Temporal Attention Encoding. First, the PSE and PE are applied to TS T . Then, the PE outputs generated on source and target SITS (i.e., pe S and pe T , respectively) are compared to re-align pe T to the representation that the PSE+TAE model adopts to discriminate crop types. Finally, the TAE generates the crop type classification for TS T considering the re-aligned target PE output, i.e., pe T .

Figure 1

 1 Figure 1 shows the schematic representation of the proposed xAI approach based on three main components: (a) SITS Encoding, (b) Positional Encoding Correction, and (c) Temporal Attention Encoding. Due to the variability in climate and cultivars, the same crop type can display different timing of its

Fig. 2 .

 2 Fig. 2. Qualitative example of PE outputs generated in the source and target domains for the same crop type (i.e., winter wheat): (a) pe T , (b) time series of NDVI on the TS T , (c) crops locations, (d) pe S , and (e) time series of NDVI computed on the TS S .

Table 2 .

 2 F1% and OA% obtained on the test set located in mid-west France (Sentinel-2 tile 30TXT) with the Standard and the Proposed PSE+TAE architecture when training with the data from: (i) Denmark (DK), (ii) Austria (AT), (iii) southern France (FR1), and (iv) the three countries (AT+DK+FR1). The best results are highlighted in bold per crop type.

	Crop Type		Standard PSE+TAE (F1%)		Proposed PSE+TAE
		DK	AT	FR1	AT+DK+FR1	DK	AT	FR1	AT+DK+FR1
	Corn	89.62 89.02 67.82	95.63	84.33 81.58 36.86	92.87
	Horsebeans	0.00	21.21 53.24	79.09	0.00	52.78	7.62	78.95
	Meadow	44.00 95.57 88.25	97.22	54.53 93.81 91.41	96.69
	Spring barley	11.95 30.77	0.72	41.23	10.47 27.37	2.62	21.95
	Unknown	21.55 48.99 38.90	68.50	23.14 43.31 37.83	62.91
	Winter barley	18.36 43.33 73.18	54.79	24.97 78.75 60.12	80.00
	Winter rapeseed	91.43 92.53 71.63	98.76	83.77 62.26 70.78	98.33
	Winter triticale	0.00	29.95 22.90	40.00	0.35	15.57	8.93	63.37
	Winter wheat	7.19	60.16 68.57	90.27	48.36 85.04 73.44	94.41
	OA%	38.18 78.97 68.94	89.63	46.26 82.33 69.17	90.42
	Macro avg. F1%	31.57 56.84 53.91	73.95	36.66 60.05 43.29	76.60
	Weighted avg. F1% 41.34 79.84 73.87	89.98	53.15 81.78 70.51	90.87
	when using the largest training dataset (AT+DK+FR1). In
	particular, the Proposed PSE+TAE is able to increase the
	OA%, macro average F1% and weighted F1% of almost 1%,
	3% and 1% compared to the Standard PSE+TAE architecture.

https://github.com/adelabbs/XAI4EO/