A Xanthomonas effector protein contributes quantitatively to virulence by inducing at least two minor Susceptibility genes

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Pérez-Quintero , Aurélie Le Ru , View ORCID Profile Caroline Bellenot , Ivanna Fuentes , View ORCID Profile Zoë E. Dubrow , View ORCID Profile Adam J. Bogdanove , View ORCID Profile Laurent D. Noël , View ORCID Profile Corinne Audran doi: https://doi.org/10.1101/2025.07.31.667891 Brice Charleux 1 LIPME, Université de Toulouse , INRAE, CNRS, Castanet-Tolosan, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Brice Charleux Carine Gris 1 LIPME, Université de Toulouse , INRAE, CNRS, Castanet-Tolosan, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sébastien Carrère 1 LIPME, Université de Toulouse , INRAE, CNRS, Castanet-Tolosan, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sébastien Carrère Alvaro L. Pérez-Quintero 2 PHIM, Université de Montpellier , IRD, CIRAD, INRAE, Institut Agro, Montpellier, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alvaro L. Pérez-Quintero Aurélie Le Ru 3 TRI-FRAIB Imaging Platform, Université de Toulouse , CNRS, Castanet-Tolosan, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Caroline Bellenot 1 LIPME, Université de Toulouse , INRAE, CNRS, Castanet-Tolosan, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Caroline Bellenot Ivanna Fuentes 1 LIPME, Université de Toulouse , INRAE, CNRS, Castanet-Tolosan, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Zoë E. Dubrow 4 Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University , Ithaca, NY, U.S.A Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Zoë E. Dubrow Adam J. Bogdanove 4 Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University , Ithaca, NY, U.S.A Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Adam J. Bogdanove Laurent D. Noël 1 LIPME, Université de Toulouse , INRAE, CNRS, Castanet-Tolosan, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Laurent D. Noël Corinne Audran 1 LIPME, Université de Toulouse , INRAE, CNRS, Castanet-Tolosan, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Corinne Audran For correspondence: corinne.audran{at}inrae.fr Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Summary Transcription Activator-Like Effector (TALE) Tal12a is widespread in strains of Xanthomonas campestris pv. campestris ( Xcc ) causing black rot on Brassica crops. We sought to determine whether and how Tal12a contributes to disease. Transcriptomic analysis of cauliflower leaves infected with Xcc strains expressing Tal12a was combined with TALE-binding element prediction and heterologous expression assays in Nicotiana benthamiana in order to identify candidate Susceptibility ( S ) genes. Artificial TALEs (arTALEs) were used to validate the contribution of candidate target genes to susceptibility. Tal12a enhances bacterial virulence and growth in cauliflower. Transcriptomic analysis revealed 380 upregulated cauliflower genes, from which nine were selected as candidate targets. Expression of sugar transporter genes BoSWEET13 and BoSWEET14c was induced though likely indirectly. Five genes were confirmed as direct targets of Tal12a. Functional assays showed that BoIAA7c auxin-dependent transcriptional regulators and BoSWEET14c each independently contribute to disease, but not to bacterial proliferation. This work identifies the first susceptibility genes in cauliflower. Tal12a enhances susceptibility in cauliflower thanks to a complex transcriptional reprogramming comprising both direct and indirect tal12a targets, some of which act as minor S genes. INTRODUCTION Phytopathogenic bacteria have evolved sophisticated molecular strategies to manipulate host physiology and establish disease. Among the virulence factors employed, Transcription Activator-Like Effector proteins (TALEs) represent a unique class of type III effectors translocated into the plant cell via the bacterial type-III secretion system ( Boch & Bonas, 2010 ). TALEs are targeted to the nucleus thanks to their C-terminal nuclear localization signals ( Yang & Gabriel, 1995 ; Szurek et al ., 2002 ) where they act as eukaryotic transcription factors by binding to DNA, and increasing the expression of host genes ( Hutin et al ., 2015a ). TALEs are deployed predominantly by Xanthomonas and Ralstonia species where they directly upregulate expression of plant genes, some of which promote colonization and infection spread and are referred to as Susceptibility ( S ) genes ( Kay & Bonas, 2009 ; Boch & Bonas, 2010 ; de Lange et al ., 2013 ). TALE proteins bind to specific plant DNA sequences through their central DNA binding domain. This domain is composed of a series of nearly identical repeats, each characterized by two critical amino acids known as Repeat-Variable Diresidues (RVDs), which define the specificity of DNA base recognition ( Moscou & Bogdanove, 2009 ; Boch et al ., 2009 ). The combination of RVDs within a TALE determines its binding to specific DNA sequences known as effector binding elements (EBEs). As a consequence, the analysis of RVD sequences using bioinformatics tools, such as TALvez, PrediTALE or TALE-NT enables the prediction of potential EBE for a given TALE within a genome ( Doyle et al ., 2012 ; Pérez-Quintero et al ., 2013 ; Erkes et al ., 2019 ). Building on this knowledge, researchers have developed artificial TALEs (arTALEs) which are engineered to recognize custom DNA sequences. These synthetic effectors have become widely used tools to specifically induce host gene expression and test the biological function of candidate genes during infection ( Weber et al ., 2011 ; Streubel et al ., 2013 ). Among the well-characterized S genes, the clade III members of the Sugars Will Eventually be Exported Transporters ( SWEET ) gene family have received considerable attention due to their targeting by various TALEs in numerous pathosystems and their key biological role during infection ( Gupta et al ., 2021 ; Baruah et al ., 2025 ). For instance, TALE-mediated transcriptional upregulation of any of the rice genes OsSWEET11, OsSWEET13 and OsSWEET14 is essential for disease symptom development and enhanced bacterial proliferation ( Yang et al ., 2006 ; Streubel et al ., 2013 ; Zhou et al ., 2015 ). Failure to induce SWEET gene expression by TALEs yields rice plants essentially resistant to Xanthomonas oryzae pv. oryzae ( Xoo ) ( Antony et al ., 2010 ; Liu et al ., 2011 ; Hutin et al ., 2015b ; Eom et al ., 2019 ; Oliva et al ., 2019 ; Schepler-Luu et al ., 2023 ). Beyond rice, TALE-mediated activation of SWEET genes has also been reported in cotton and cassava, indicating that the exploitation of SWEET genes represents a convergent strategy employed by various Xanthomonas species to facilitate infection ( Cohn et al ., 2014 ; Cox et al ., 2017 ). Other examples of S genes activated by TALEs include OsSULTR3;6 , a putative sulfate transporter gene associated with susceptibility to bacterial leaf streak in rice ( Cernadas et al ., 2014 ). CsLOB1 , a transcription factor important bacterial canker symptoms on citrus, is targeted by several TALEs from Xanthomonas citri , ( Hu et al ., 2014 ). In wheat, the TaNCED-5BS gene, which encodes a key enzyme involved in abscisic acid biosynthesis, has been identified as a S gene by inhibiting plant defense during bacterial streak disease ( Peng et al ., 2019 ). Last but not least, several examples of transcription factors targeted by TALEs have been reported to contribute to susceptibility in rice such as OsTFX1 and OsERF123 ( Sugio et al ., 2007 ; Tran et al ., 2018 ). Black rot, caused by Xanthomonas campestris pv. campestris ( Xcc ), is a major vascular and seed-transmitted disease that affects all cultivated brassica crops worldwide ( Vicente & Holub, 2013 ; Dubrow & Bogdanove, 2021 ). After infection of hydathodes at the leaf margins, Xcc spreads through the xylem vessels and colonizes the mesophyll ( Vicente & Holub, 2013 ; An et al ., 2020 ). Unlike other Xanthomonas species, the presence of tal genes in pathogenic Xcc strains is highly variable, with some strains harboring several TALEs, while others are completely devoid of them. A recent study demonstrated that inhibition of tal genes by CRISPR interference (CRISPRi) in the Xcc strain CN08 resulted in decreased disease symptoms in cauliflower ( Zárate-Chaves et al ., 2023 ). Interestingly, a reduction in Xcc pathogenicity was also reported in the cauliflower pathosystem with the double deletion mutant Δ tal12a/15a in strain Xca5 ( Denancé et al ., 2018 ). Among the TALEs identified, Tal12a, which comprises 12 RVDs, is the most widespread TALE effector in an Xcc diversity panel of 23 strains ( Denancé et al ., 2018 ). The present study aimed to determine the contribution of Tal12a to Xcc virulence in cauliflower and to identify its host targets. We showed that Tal12a enhances bacterial pathogenicity and in planta proliferation. We identified 380 candidate target genes of Tal12a in cauliflower by a transcriptomic approach. Among these, five were confirmed as direct targets. We demonstrated that BoSWEET14c and BoIAA7c play a role in host susceptibility. These findings suggest that Tal12a manipulates both cauliflower auxin signaling and sugar transport pathways to promote disease susceptibility in cauliflower. MATERIALS AND METHODS Plant material Cauliflower plants ( Brassica oleracea cv. botrytis var. Clovis F1, Vilmorin) were grown in a greenhouse. Nicotiana benthamiana plants were sown on soil and grown at 24°C under long-day photoperiod (16-h light) with 60% relative humidity. Four-to-five weeks-old plants were used. Bacterial strains, plasmids and growth conditions All bacterial strains used in this study are described in Table S1 . Xcc strains were grown either in MOKA medium (yeast extract 4g/L, casamino acids 8g/L, K 2 HPO 4 2g/L, MgSO 4 .7H 2 O 0.3g/L) or in MME medium (K 2 HPO 4 10.5 g/L, KH 2 PO 4 4.5 g/L, (NH 4 ) 2 SO 4 1 g/L, casamino acids 1.5g/L, MgSO 4 ,7H 2 O 0.3g/L) with the appropriate antibiotics at 28°C. Agrobacterium tumefaciens GV3101 strains were grown in YEB medium (yeast extract 1g/L, beef extract 5g/L, peptone 5g/L, sucrose 5g/L, MgSO 4 0.4mM pH7.2) with the appropriate antibiotics at 28°C. CN08 wild type strain was isolated by He et al ., (2007) . Rifampicin-resistant and tal12a mutant derivatives of CN08 were obtained spontaneously. CN08Δ tal12a was complemented with tal12a cloned in pSKX1 ( Tran et al ., 2018 ). The Δ26 polymutant strain derivatives of Xcc strain 8004 lacks to coding regions of 26 type III effectors or secreted proteins (european patent EP20305940.7): XopAC, AvrBs1, XopH, XopX1, XopX2, XopF, HrpW, XopD, XopAN, XopQ, XopK, XopJ5, XopG, AvrXccA1, AvrXccA2, XopAM, XopE2, XopAH, XopR, XopL, XopZ, XopAG, XopP, XopAL1, XopAL2 and XopN . TALE cloning vectors were created as described in Mormile et al . (2025) . tal genes were integrated in the Δ26 strain genome between XC_3385 and XC_3386 genes as described in Luneau et al . (2022) . TALE protein accumulation in all strains was assessed by Western blot analysis using a polyclonal antibody developed in rabbit against an Xcc TALE ( Zárate-Chaves et al ., 2023 ). Rifampicin (50µg/mL), kanamicin (50µg/mL) and gentamicin (10 and 15µg/mL for Xanthomonas and Agrobacterium , respectively) were used for antibiotic concentrations. Virulence and in planta growth assays on cauliflower Pathogenicity assays were conducted on the second true leaf by piercing the central vein using a needle dipped in a bacterial suspension at 10 8 CFU/mL. Three to five independent biological repetitions were performed with five to six plants each time. Disease development was scored visually at 4, 7 or 10 dpi, with a disease index scale from 0 (no symptom) to 4 (V-shape like leaf necrosis), as described ( Denancé et al ., 2018 ). For virulence assays, mesophyll infiltration was conducted on the second true leaf using a needleless syringe filled with a bacterial suspension at 10 8 CFU/mL. At least three independent biological repetitions were performed with three plants each time. Symptoms were observed between 7 and 8 dpi. In planta bacterial populations were determined after mesophyll infiltration of the second true leaf using a syringe filled with a bacterial suspension at 10 6 CFU/mL. One leaf disc (6 mm diameter) was harvested at 0 and 3 dpi and ground with glass beads in 200µL 1mM MgCl 2 . The homogenate was serially diluted and spotted three times on MOKA plates supplemented with pimaricin (30µg/mL) and appropriate antibiotics. Plates were incubated at 28°C for 48h and colonies enumerated in spots containing 1 to 30 colonies. Bacterial densities in leaves were expressed as log CFU/cm 2 . Four independent biological repetitions were performed with six plants. In vitro bacterial growth assay Bacterial growth of Xcc strains was monitored in MOKA and MME media by measuring optical density à 600nm over 24 hours. Growth rates were calculated during the exponential phase as described ( Lauber et al ., 2024 ). Three independent biological replicates were performed. Tissue sampling, RNA extraction and transcriptomic analyses Mesophyll infiltrations were carried out on the second leaf of the plant, following previously described methods, using a bacterial suspension at 10⁸ CFU/mL. Fifteen leaf discs (7-mm diameter) were collected on three plants (five per plant) at 1 dpi and immediately frozen in liquid nitrogen. Three independent biological replicates were performed. RNA was extracted using either the Macherey-Nagel NucleoSpin RNA Plus Kit or the Qiagen RNeasy Mini Plant Kit, following the manufacturer’s protocols. Global gene expression profiles were determined by oriented paired-end RNA sequencing (2x 150 bp) on an Illumina NovaSeq 6000 using the Illumina TruSeq Stranded mRNA Kit. Raw paired-end reads were processed using the nf-core/rnaseq pipeline version 3.0 (doi:10.5281/zenodo.4323183). We used the pseudo-mapping strategy with salmon software (version 1.4.0) and reference genome annotation of Brassica oleracea ( https://ftp.ensemblgenomes.ebi.ac.uk/pub/plants/release-57/gff3/brassica_oleracea ). Counts were retrieved at the gene level and were normalized using the EdgeR package and the CPM/TMM method ( Robinson & Oshlack, 2010 ). After quality control, differential analysis was performed using fitted generalized linear models (GLMs) with a design matrix multiple factors (biological repetition and factor of interest). DEGs were called using the GLM likelihood ratio test using a false discovery rate (FDR Benjamini-Hochberg) adjusted p value < 0.01. GOterm were associated with the genes using Blast2GO ( Conesa et al ., 2005 ). Analysis of GO enrichment was conducted using the topGO package v. 2.52.0 ( Alexa et al ., 2006 ). For quantitative real-time-PCR (qRT-PCR) analyses, DNase treatment was carried out using the Invitrogen™ TURBO DNA-free™ Kit, and cDNA was generated from 1µg RNA using the Transcriptor Reverse Transcriptase Kit by Roche. qRT-PCR was then conducted using the Takyon™ No ROX SYBR 2X MasterMix Blue dTTP Kit, following the manufacturer’s protocol. Primers for qRT-PCR used in this study are listed in Table S2 . TALE target prediction The promoter regions (1000 nt upstream the translation start site) of annotated genes in TO1000 were extracted from B. oleracea genome (see above) using the GenomicRanges package in R ( Aboyoun et al ., 2013 ). Predictions of binding sites were made using Talvez v3.2 ( Pérez-Quintero et al ., 2013 ) and Preditale ( Erkes et al ., 2019 ), the top 300 predicted targets according to score were kept for each RVD sequence. Promoter sequence cloning and transient co-expression assays in N. benthamiana Promoter sequences were amplified using primers with attB sites (Table S2 ) and verified by sequencing (Table S3 ). The sequences were cloned into the pDONR207 vector using Gateway™ BP Clonase™ II Enzyme mix and transferred to the pJawohl11-GW-gus destination vector using Gateway™ LR Clonase™ II Enzyme mix, following the manufacturer’s instructions. The resulting plasmids were electroporated into Agrobacterium tumefaciens GV3101. For co-transient expression in N . benthamiana , A. tumefaciens and Xanthomonas strains were grown and resuspended in infiltration medium (10 mM MES pH 5.6, 10 mM MgCl 2 , 150 μM acetosyringone) to OD 600nm = 0.3 and OD 600nm = 0.1, respectively. After a 2 hours incubation at room temperature, the bacterial suspensions were infiltrated into the leaves of four-week-old N. benthamiana plants using a needleless syringe. Plants were incubated for two days in growth chambers. To assess GUS activity, 12 mm leaf discs were harvested from the inoculated leaves and vacuum infiltrated with a substrate-detergent solution containing 1 mM 5-bromo-4-chloro-3-indolyl β-D-glucuronide, 0.2% Triton X-100 [v/v], 2 mM K 3 FeCN 6 , 2 mM K 4 FeCN 6 , and 50 mM sodium phosphate buffer, pH 7.2. The discs were incubated overnight at 37°C, then fixed and destained in 80% ethanol. GUS staining intensity was quantified using ImageJ software. The experiment included four independent biological replicates, each consisting of three plants. RESULTS tal12a contributes to the virulence of Xcc in cauliflower To investigate the biological importance of tal12a in the CN08 strain, we evaluated the pathogenicity of a spontaneous mutant lacking this gene (CN08Δ tal12a ) and a complemented strain carrying tal12a on the pSKX1 vector (CN08Δ tal12a pSKX1- tal12a ). Western blot analysis confirmed the absence/presence of the 12-repeat-long TALE corresponding to Tal12a in strains CN08Δ tal12a and CN08Δ tal12a pSKX1- tal12a , respectively while the expression of the other three TALEs (15-, 18- and 22-repeat-long TALEs) remained unchanged (Fig. S1a ). Xcc strains were wound-inoculated in the leaf midvein of susceptible cauliflower (cv. Clovis) and symptoms were monitored at four and seven days post-inoculation (dpi) ( Fig. 1a,b ). The CN08Δ tal12a mutant caused significantly less chlorosis and necrosis on cauliflower than the wild type. Symptom development was fully restored upon complementation with tal12a , demonstrating Tal12a contribution to the virulence of strain CN08. Download figure Open in new tab Fig. 1: tal12a promotes bacterial virulence and proliferation on cauliflower. ( a ) Boxplot representation of disease symptoms caused by Xcc CN08 strain at 4 and 10 days post inoculation (dpi), respectively. Pathogenicity assays were performed using the WT Xcc strain CN08, its tal12a deletion mutant CN08Δ tal12a and the mutant complemented with either the empty vector pSKX1 (+EV) or the tal12a gene (+ tal12a ). ( b ) Representative photographs for the median disease symptoms caused by Xcc CN08 strain derivatives at 7 dpi. ( c ) Boxplot representation of disease symptoms caused by Xcc at 10 dpi. Pathogenicity was evaluated in the WT Xcc strain 8004 or its Δ26 polymutant derivative, which lacks 26 genes encoding type III effectors, either alone or complemented with tal12a or tal14c integrated into the genome. ( d ) Xcc Δ26 strain titers in cauliflower leaves were quantified at 0 and 3 days after mesophyll infiltration with a bacterial suspension at 10 6 CFU.mL −1 . Results represent four independent experiments with six plants per strain. Statistical groups were determined using the nonparametric Kruskal–Wallis test (p < 0.05) and are indicated by different letters. ( a-c ) Cauliflower leaves (cv. Clovis) were wound inoculated by piercing the central vein with a needle dipped in a bacterial suspension at 10 8 CFU.mL −1 . The disease index was as follows: 0, no symptoms; 1, weak chlorosis around wounded sites; 2, strong chlorosis; 3, first necrotic symptoms; 4, large necrotic lesions as described in Luneau et al . (2022) . Results correspond to three independent experiments with at least six plants per strain. The statistical groups were determined using the nonparametric Kruskal–Wallis test (p < 0.05) and are indicated by different letters. To specifically assess the contribution of tal12a in Xcc virulence on cauliflower by a gain-of-function experiment, we used a disarmed derivative of Xcc strain 8004 named 8004Δ26T3E (hereafter referred to as Δ26; European patent EP20305940.7). This polymutant strain possesses a functional type III secretion system,naturally lacks endogenous tal genes, and has been engineered through the successive deletion of twenty-six genes encoding type-III effector proteins. The Δ26:: tal12a and Δ26:: tal14c strains harbor a stable genomic integration of tal12a or tal14c , a 14-repeat tal gene used as a control to assess the specificity and function of tal12a targets ( Denancé et al ., 2018 ). Expression of the Tal12a and Tal14c proteins was confirmed by Western blot analysis in the Δ26:: tal12a and Δ26:: tal14c strains, respectively (Fig. S1b ). These two strains showed no detectable fitness deficit in vitro relative to the parental Δ26 strain, as measured by maximum growth rates (µmax) in MME and MOKA culture media (Fig. S2 ). At 10 dpi, the Δ26 strain failed to induce disease symptoms, similar to the mock-inoculated plants, the wild-type 8004 strain caused severe chlorotic and necrotic symptoms ( Fig. 1c ). Notably, Δ26 strains expressing either tal12a or tal14c exhibited an increase in disease severity compared to the Δ26 strain ( p<0.01 ), indicating that each TALE can partially restore virulence. To further evaluate their contribution to bacterial proliferation, bacterial titers were measured at 3 dpi ( Fig. 1d ). A significant increase in bacterial growth was observed for the Δ26:: tal12a strain but not Δ26:: tal14c compared to Δ26 ( Fig. 1d ). Together, these results establish tal12a as a virulence factor of Xcc , enhancing both symptom development and bacterial colonization in cauliflower. Tal12a increases directly or indirectly the expression of 380 cauliflower genes To identify the cauliflower genes whose expression is specifically activated by Tal12a, we conducted a transcriptomic analysis by RNA sequencing on cauliflower leaves infected with the Δ26:: tal12a strain and compared them to leaves infected with the Δ26 strain at 1 dpi. The Δ26:: tal14c strain was included to assess the specificity and function of Tal12a targets. The properties of the RNAseq libraries are given in Table S4 . As expected from transcriptional activators, only a limited number of downregulated genes were identified following infection with strains Δ26:: tal12a (10) and Δ26:: tal14c (38), suggesting these are likely the result of indirect TALE-dependent regulations ( Fig. 2a ). Expression of 380 and 713 genes was upregulated (fold change ≥ 2; FDR < 0.01) by Δ26:: tal12a and Δ26:: tal14c , respectively ( Fig. 2a ). Among these, 43 genes were induced by both TALEs. A hypergeometric test confirmed that this overlap was statistically significant (p-value < 0.001) and unlikely to occur by chance. Gene Ontology (GO) enrichment analysis of these 43 genes revealed a strong and significant enrichment in categories related to “jasmonic acid” (JA), “wound responses” and “regulation of defense responses” ( Fig. 2c ). These results suggest that TALEs may modulate disease-associated stress responses by converging directly or indirectly on shared downstream pathways. Download figure Open in new tab Fig. 2: Tal12a induces expression of 380 cauliflower genes. Transcriptomic analysis was performed on cauliflower leaves infected by Δ26 strain derivatives expressing either Tal12a (Δ26:: tal12a ) or Tal14c (Δ26:: tal14c ) at 1 dpi and compared to strain Δ26. ( a ) Venn diagram showing genes whose expression is regulated by Tal12a and Tal14c (FDR 2). ( b,c ) Gene ontology (GO) analysis highlights the biological processes enriched among genes regulated by both tal12a and tal14c ( b ) and by tal12a only ( c ). The x -axis represents the enrichment probability of a given GO term. The size of the circle represents gene count. The color scale represents the enrichment of the GO term in the up-regulated gene list compared to the background population of genes. This convergence could result from the activation of common target genes, functionally redundant paralogues, or from plant perception mechanisms that respond similarly to both TALEs, independently of their DNA-binding specificities and target gene identities. GO-term enrichment analysis for genes specifically upregulated by Tal12a revealed distinct biological processes compared to those associated with Tal14c ( Fig. 2b , Table S5 ). GO terms such as "megagametogenesis", “ABA metabolic pathway”, “pollen development”, and cell-wall-related categories (“xyloglucan metabolic process”, “pollen development”, “hexose metabolic process”, “cell wall biogenesis”) were significantly enriched among Tal12a-regulated genes, indicating that Tal12a impacts physiological pathways distinct from Tal14c. Notably, the term “pollen development” was associated with the expression of two clade III SWEET genes, BoSWEET13 and BoSWEET14c (Zhang et al., 2019). Interestingly, BoSWEET13 is the most upregulated (greatest fold-change) gene in the transcriptomic analysis when comparing Δ26:: tal12a to Δ26 ( Table 1 ). These genes are homologous to OsSWEET13 and OsSWEET14 in rice, which are known S genes induced by TALEs such as PthXo2, PthXo3, AvrXa7, TalC and TalF from Xoo ( Teper et al., 2023 ). SWEET genes are thus obvious S gene candidates to mediate the virulence function of Tal12a in cauliflower. View this table: View inline View popup Download powerpoint Table 1: Cauliflower genes induced by Tal12a at 1 dpi and selected for downstream analysis A set of 29 genes identified as candidate direct targets of Tal12a in cauliflower In order to identify direct targets of Tal12a and Tal14c in cauliflower, promoters of genes significantly upregulated (FDR < 0.01) by Tal12a and Tal14a, respectively, were inspected for predicted effector binding elements (EBE) using TALVEZ and PrediTALE ( Pérez-Quintero et al ., 2013 ; Erkes et al ., 2019 ). We restricted our analysis to the 300 predictions with the highest EBE scores for each TALE. Among the 713 genes upregulated by Tal14c, only 20 harbored high-score EBE predictions within their promoter regions, as identified by at least one of the two prediction tools (Fig. S3 , Table S6 ). In the case of Tal12a, 29 out of 380 upregulated genes were identified as potential direct targets based on EBE predictions ( Fig. 3a , Table S6 ). Notably, the 29 candidate direct targets of Tal12a did not overlap with those identified for Tal14c. This list was refined using the features often found in direct TALE targets: (i) high EBE prediction scores, (ii) close proximity of EBEs to the TATA box, (iii) strong transcriptional induction. Considering gene annotations, seven candidate genes were prioritized for further characterization ( Table 1 and Table S6 ). These genes encode a cytochrome P450 gene (Bo3g023170), a translocon protein TIC40 located on the inner chloroplast membrane (Bo2g013210) and five genes encoding transcription factors or related proteins ( ERF043 (Bo3g171090), ATHB-X (Bo6g112570), and three IAA7 isoforms (Bo1g103470, Bo3g081600, and Bo5g093110). In addition, given the well-established role of SWEET genes as major S genes in several pathosystems and their direct activation by TALE ( Streubel et al ., 2013 ; Cohn et al ., 2014 ; Teper et al ., 2023 ), we also studied BoSWEET13 (Bo9g097170) and BoSWEET14c (Bo7g109890). Although no high-confidence EBE was predicted in their promoter regions, their expression was highly and specifically upregulated by Tal12, suggesting that their induction by Tal12a may occur through an indirect regulatory mechanism. Download figure Open in new tab Fig. 3: Tal12a specifically modulates the expression of cauliflower genes. ( a ) Venn diagram showing the intersections between genes differentially expressed in response to Tal12a (FDR2) and genes predicted to contain a Tal12a EBE using the PrediTALE and TALVEZ tools. The top 300 predicted targets were retained for comparison ( Pérez-Quintero et al ., 2013 ; Erkes et al ., 2019 ). ( b ) Gene expression of nine putative Tal12a target genes following inoculation: BoCYP450 , BoERF043 , BoIAA7a , BoIAA7b , BoIAA7c , BoTIC40 , BoSWEET13 , BoSWEET14c and BoATHB-X . Cauliflower leaves were infected with various Xcc strains derivatives and sampled one day post-inoculation. The CN08Δ tal12a mutant was transformed with either the empty vector pSKX1 (EV) or with tal12a (+ tal12a ). The Δ26 polymutant strain and its derivatives expressing tal12a or tal14c (Δ26:: tal12a or Δ26:: tal14c , respectively) were inoculated. Each strain was tested in three independent experiments, each comprising three plants. Gene expression was measured by qRT-PCR. Data points represent at least eight measurements per condition. Statistical analysis was performed using a Wilcoxon test, comparing CN08 WT with its derivatives and Δ26 with its derivatives (*: p<0.05; **: p<0.01; ***: p<0.001). To independently validate the induction of these genes by Tal12a, we compared their expression levels in cauliflower by qRT-PCR following inoculation with Xcc strains expressing or not expressing tal12a . Gene expression analysis at 1 dpi revealed that introducing tal12a in the Δ26 polymutant strain significantly upregulated all seven candidate genes as well as the two SWEET genes, corroborating the results obtained in the previous transcriptomic analysis ( Fig. 3b ). We also tested strains CN08 and Xca5 silenced for tal gene expression using a CRISPRi strategy targeting the conserved ribosome binding site (RBS) of tal genes as described ( Zárate-Chaves et al ., 2023 ). CN08 and Xca5 express four and three tal genes, respectively, and share both tal12a and tal22a ( Denancé et al ., 2018 ). Compared to both wild-type (WT) CN08 and control (Ctl) strains, eight out of nine Tal12a target genes showed a statistically significant decrease in expression in the CN08 CRISPRi-RBS strain relative to the wildtype and control strains ( Fig. 4 ). Similarly, expression of five out of nine target genes was significantly reduced in the Xca5 CRISPRi-RBS strain relative to WT Xca5 and control strains. These results demonstrate the TALE-dependent regulation of the nine candidate genes with some strain to strain variations. To determine the specific contribution of Tal12a, expression of the nine genes was tested in a CN08Δ tal12a mutant. This deletion resulted in a statistically significant reduction in expression of the BoCYP450 , BoERF043 , BoIAA7a , BoIAA7b , BoIAA7c , BoSWEET13 , BoSWEET14c , and BoATHB-X genes, while the expression of BoTIC40 remained unaffected ( Fig. 3b ). Expression levels were restored to wild-type levels upon complementation with a plasmid carrying tal12a ( Fig. 3b ). These various results obtained with different Xcc strains confirm that the expression of the eight selected genes is promoted by Tal12a, either directly or indirectly. The subtle differences observed between strains may result from incomplete CRISPRi-mediated silencing or from variations in TALE accumulation levels. Download figure Open in new tab Fig. 4: Silencing of tal12a in Xcc modulates the expression of potential target genes in cauliflower. Gene expression of nine potential Tal12a target genes ( BoCYP450 , BoERF043 , BoIAA7a , BoIAA7b , BoIAA7c , BoTIC40 , BoSWEET13 , BoSWEET14c , and BoATHB-X ). Cauliflower leaves were infected with various Xcc strains expressing or not Tal12a and ampled at 1 dpi. CN08 and Xca5 CRISPRi (RBS) and control (Ctl) strains are derivatives of the wild-type (WT) CN08 and WT Xca5 ( Zárate-Chaves et al ., 2023 ). Each strain was tested in three independent experiments, each involving three plants. Data points represent at least eight measurements per condition. Statistical analysis was performed using a Wilcoxon test, with significance set at p<0.05, comparing WT with CRISPRi derivatives. Both direct and indirect regulation of Tal12a targets was revealed by transactivation assays in N. benthamiana In order to assay a direct regulation of the nine genes by Tal12a, we placed their promoter sequences (307 to 920 bp including the predicted EBE when present, Table S3 ) upstream of promoterless gus reporter gene. These fusions were transiently delivered in N. benthamiana using Agrobacterium tumefaciens while Tal12a was delivered using the Xcc Δ26:: tal12a strain. Transactivation of the gus gene expression was assessed by quantifying GUS activity on leaves 2 dpi using ImageJ ( Fig. 5 ). Basal GUS activity was negligible for the BoCYP450 , BoERF043 , BoIAA7a , BoIAA7b , BoTIC40 , and BoATHB-X promoter constructs in absence of Tal12a. A significant increase in GUS activity was observed upon co-delivery with Δ26:: tal12a , relative to the Δ26 strain alone. These results support the EBE prediction and suggest direct regulation by Tal12a for BoCYP450 , BoERF043 , BoIAA7a , BoIAA7b , BoTIC40 , and BoATHB-X . Differences in GUS activity were not statistically significant for the BoIAA7c , BoSWEET13 and BoSWEET14c constructs consistent with the absence of predicted EBE for BoSWEET13 and BoSWEET14c. These results indicate that Tal12a-mediated upregulation of BoSWEET13 and BoSWEET14c is indirect. Download figure Open in new tab Fig. 5: Tal12a-mediated transcriptional transactivation of candidate cauliflower susceptibility gene promoters in N. benthamiana . Promoter regions of nine Tal12a target genes ( BoCYP450 , BoERF043 , BoIAA7a , BoIAA7b , BoIAA7c , BoTIC40 , BoSWEET13 , BoSWEET14c , and BoATHB-X ) were fused to a gus reporter gene and delivered into N. benthamiana leaves via Agrobacterium -mediated transformation, either alone or in co-infiltration with the Xcc Δ26:: tal12a strain or the Δ26 strain. GUS expression levels were quantified at 2 dpi using ImageJ. The experiment was independently repeated four times, with three plants per experiment, resulting in at least 11 data points per condition. Statistical significance was assessed using a Wilcoxon test, comparing the Δ26:: tal12a condition to the Δ26 control for each promoter (*: p<0.05; **: p<0.01; ***: p<0.001) BoIAA7c and BoSWEET14c are minor S genes promoting disease symptoms in cauliflower We assessed the contributions of BoIAA7c , BoSWEET13 , and BoSWEET14c in cauliflower susceptibility to Xcc by asking whether their individual transcriptional activation by artificial TALEs (arTALEs) could recapitulate Tal12a virulence functions. Two arTALEs were designed per promoter region ( Fig. 6a ) and introduced into the Δ26 Xcc strain. In order to test for arTALE functionality, BoIAA7c , BoSWEET13 , and BoSWEET14c transcript levels were quantified by qRT-PCR at 1 dpi following inoculation with Δ26 strains expressing the respective arTALE. As expected, expression of tal12a from a plasmid into the Δ26 strain (Δ26+Tal12a) significantly increased the expression of all three candidate genes compared to the Δ26 carrying the empty vector (Δ26+EV) ( Fig. 6b ). Similarly, each arTALE construct designed to specifically target BoIAA7c , BoSWEET13 , or BoSWEET14c upregulated its target gene, reaching or even exceeding the levels observed with Tal12a ( Fig. 6b ). Interestingly, arTALE13.2 also induced expression of BoSWEET14c in contradiction with in silico EBE predictions. To investigate the impact of arTALE-mediated activation of those three genes on disease development and bacterial fitness in planta , Xcc strains were infiltrated in the mesophyll of cauliflower leaves. While the Δ26+empty vector strain caused only mild chlorosis at 7 dpi, strain Δ26+Tal12a caused stronger chlorosis and necrosis revealing Tal12a virulence functions in this tissue ( Fig. 6c , Fig. S4 ). Δ26 strains expressing arTALE7.1, arTALE7.2, arTALE13.2, arTALE14.1, or arTALE14.2 also triggered more severe disease symptoms compared to the EV control or Δ26+arTALE13.1. These results indicate that BoIAA7c and BoSWEET14c but not BoSWEET13 contribute to symptom development on cauliflower. Yet, no significant increase in bacterial proliferation could be observed at 3 dpi with any of the arTALEs, in contrast with strains expressing Tal12a ( Fig. 6d ). These findings suggest that BoIAA7c and BoSWEET14c contribute to the virulence-promoting function of Tal12a in Xcc -cauliflower interactions and function as minor S genes in this pathosystem. Download figure Open in new tab Fig. 6: Tal12a and arTALE inducing expression of BoIAA7c and BoSWEET14c expression promote symptom development in cauliflower mesophyll. ( a ) ArTALE target sites designed based on the promoter sequences of BoIAA7c , BoSWEET13 and BoSWEET14c . ( b ) Expression of BoIAA7c , BoSWEET13 and BoSWEET14c were quantified by qRT-PCR in cauliflower leaves infiltrated with the Δ26 strain carrying either the empty vector pSKX1 (EV), the tal12a gene (+Tal12a) or individual arTALE constructs at 10⁸ CFU·mL - ¹ for each strain. Samples were collected at 1 dpi. The experiment was repeated three times independently, using three plants per replicate, yielding at least nine biological data points per condition. ( c ) Representative images showing the median disease symptoms at 7 dpi following infection with the Xcc Δ26 strain complemented with either the empty vector pSKX1 (EV), tal12a (+Tal12a) or arTALEs. Inoculations were performed on cauliflower leaves by syringe infiltration with bacterial suspensions at 10⁸ CFU·mL - ¹. Each condition was tested in three independent experiments using three plants per strain (Fig. S4 ). ( d ) Xcc titers in cauliflower leaves were measured at 0 and 3 dpi following mesophyll infiltration with a bacterial suspension at 10 6 CFU.mL −1 . Three independent experiments were performed, each involving six plants per strain. Statistical significance in panels ( b ) and ( d ) was assessed using a Wilcoxon test, comparing the different strain against the Δ26 + empty vector (EV) control (*: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0,0001). In order to explore a possible synergy between these two S genes both activated by Tal12a, we co-delivered a 1:1 mix of Δ26 strains expressing arTALEIAA7.1 and either arTALE13.1, arTALE13.2, or arTALE14.2. arTAL-mediated induction of the expected genes was confirmed for each combination ( Fig. 7a ). Co-expression of BoIAA7c and BoSWEET14c, achieved upon arTALE7.1/arTALE14.2 co-delivery, was consistently associated with increased symptom severity relative to the empty vector. In contrast, co-expression of BoIAA7c and BoSWEET13 (arTALEIAA7.1/arTALE13.1) had a milder effect on symptom severity ( Fig. 7b , Fig. S5 ). Interestingly, co-inoculation with arTALEIAA7.1 and arTALE13.2, which induced BoIAA7c , BoSWEET13 , and BoSWEET14c simultaneously, triggered increased symptoms in cauliflower leaves compared to the empty vector. These results support the hypothesis that BoSWEET14c and BoIAA7 function as minor S genes, and that their co-activation has an additive effect on disease symptom severity. Download figure Open in new tab Fig. 7: Co-induction of BoIAA7c and BoSWEET14c genes by a 1:1 arTALEs mix enhances disease symptom development in cauliflower mesophyll. ( a ) Gene expression of BoIAA7c , BoSWEET13 and BoSWEET14c was assessed in the mesophyll one day after infiltration with Xcc suspensions (10 8 CFU.mL −1 ) of Δ26 derivatives carrying either the empty vector pSKX1 (EV), the tal12a gene (+Tal12a) or a 1:1 mix of arTALE constructs. The experiment was repeated three times, with three plants per experiment, resulting in a minimum of 9 data points. Statistical significance were assessed using a Wilcoxon test, comparing the different strain against the Δ26 + empty vector control (*: p<0.05; **: p<0.01; ***: p<0.001). ( b ) Representative pictures of the disease symptoms caused at 7 dpi by the Δ26 strain carrying either the empty vector pSKX1 (EV), the tal12a gene (+Tal12a) or a 1:1 mix of arTALE constructs. Four independent experiments were performed with at least one plant per strain (Fig. S5 ). Xcc strains were inoculated by mesophyll infiltration of a cauliflower leaf (cv. Clovis) with a syringe filled in a bacterial suspension at 10 8 CFU.mL −1 . DISCUSSION A detectable contribution of Tal12a to Xcc virulence on cauliflower The first comprehensive analysis on Xcc tal genes was conducted in 2018 x and identified Tal12a as a predominant TALE protein present in nine out of 38 strains representative of Xcc genetic diversity ( Denancé et al ., 2018 ). Tal12a contribution to virulence was previously shown only in combination with Tal15a or Tal22a on radish ( Kay et al ., 2005 ) and with Tal15a on cauliflower ( Denancé et al ., 2018 ). However, both studies failed to show any stand-alone Tal12a virulence functions. Here, we demonstrate that Tal12a alone promotes both bacterial proliferation and disease symptom development on cauliflower by gain-of-function in the Xcc strain 8004Δ26 as well as symptom development on cauliflower by loss-of-function in strain CN08. In contrast, Tal14c enhanced symptom development without affecting bacterial growth. These results thus illustrate that Xcc tal genes likely function as minor virulence determinants in contrast to other pathogens where major tal virulence functions have long been identified such as in Xanthomonas oryzae pv. oryzae , Xanthomonas oryae pv. oryzicola , Xanthomonas citri pv. citri , Xanthomonas axonopodis pv. manihotis , Xanthomonas phaseoli pv. manihotis ( Streubel et al ., 2013 ; Cernadas et al ., 2014 ; Cohn et al ., 2014 ; Hu et al ., 2014 ; Cox et al ., 2017 ). In Xcc , virulence seems to rely primarily on non-TALE Xop type III effectors while tal genes play minor yet significant roles in virulence. Tal12a and Tal14c directly upregulate distinct target gene repertoires that converge indirectly on some shared transcriptional responses Our transcriptomic analysis revealed that Tal12a alters processes beyond classical defense, including developmental and hormonal pathways such as megagametogenesis, ABA metabolism, and pollen development suggesting a specific effect of Tal12a different from Tal14c. Despite upregulating a distinct repertoire of direct target genes, Tal12a and Tal14c induce expression of a shared set of 43 genes, none of which appear to be direct targets of Tal12a. This overlap likely reflects indirect regulation through convergent downstream signaling pathways or common stress responses. Notably, GO enrichment analysis of these shared genes reveals categories associated with jasmonic acid signaling, wounding, and regulation of defense responses. This could reflect a case of functional convergence, where different TALEs manipulate distinct upstream regulators that ultimately feed into similar host processes. This scenario is reminiscent of TALEs within a single Xoo strain exhibiting functional redundancy by targeting the same genes or host pathways ( Streubel et al ., 2013 ; Doucouré et al ., 2018 ). BoIAA7c and BoSWEET14c are minor S genes in cauliflower Considering that Tal12a consists of only twelve repeats, it is theoretically more prone to bind and induce a larger number of genes. However, there were only 29 potential direct targets, both upregulated in the presence of Tal12a and harboring a candidate EBE in the promoter region. Among these, only one or few may act as S genes mediating Tal12a virulence functions, while others could be off-targets. We also identified two SWEET genes as indirect Tal12a targets. This contrasts with other cases of TALE-mediated SWEET induction in rice, cassava and cotton where activation is direct, via an EBE in the promoter ( Zhang et al ., 2022 ). Notably, TALE-independent induction of SWEET gene expression has also been reported upon infection by Pseudomonas , Botrytis and Sinorhizobium ( Chen et al ., 2010 ). The mechanisms by which these microbes naturally deprived of tal genes induce SWEET expression have not been characterized. These findings highlight the fundamental utility of SWEET gene upregulation as a common parasitic/symbiotic strategy. Our findings suggest that Xcc may have evolved an alternative TALE-dependent mechanism to induce SWEET gene expression. We demonstrated that both BoIAA7c and BoSWEET14c function as minor S genes, contributing to symptom development without significantly increasing bacterial growth. In contrast to BoSWEET14c , BoSWEET13 did not significantly increase susceptibility, suggesting functional differences despite sequence similarities. As for BoIAA7c , its precise role in disease progression remains obscure though auxin signaling is known to facilitate bacterial infection ( Kunkel & Johnson, 2021 ). Interestingly the Pseudomonas syringae type III effector AvrRpt2 promotes the degradation of IAA27/AXR2 in Arabidopsis, thereby enhancing auxin signaling and suppressing plant immunity to facilitate infection ( Cui et al ., 2013 ). Thus, BoIAA7c and BoSWEET14c act as minor S genes, contributing quantitatively to susceptibility to Xcc in cauliflower, these genes might represent a useful resource for breeding durable resistance by loss-of-susceptibility. BoIAA7c and BoSWEET14c function additively to mediate Tal12a virulence functions in cauliflower The individual induction of BoIAA7c and BoSWEET14c expression using arTALEs enhanced disease symptoms, including necrosis, without promoting bacterial proliferation. This indicates that neither gene accounts for virulence activity of Tal12a alone. This finding is noteworthy, as it diverges from the “one TALE/one S gene” model commonly described in Xanthomonas – plant interactions, in which the activation of a single S gene is typically necessary and sufficient to trigger strong disease phenotypes. A recent exception to this paradigm is Tal7b from Xanthomonas citri pv. malvacearum , which induces multiple functionally distinct targets ( GhSWEET14a , GhSWEET14b , and GhPL1 ) that act additively to promote lesion development in cotton ( Mormile et al ., 2025 ). Similarly, our results suggest that Tal12a may act through a network of multiple S genes with complementary roles, pointing to a more complex and integrated virulence strategy than previously recognized. This observation raises the possibility that many uncharacterized TALEs contribute quantitatively to pathogenicity, acting in concert with other type III effectors to establish virulence. By elucidating these mechanisms, we pave the way for novel approaches to managing black rot disease in cruciferous crops, through the disruption of Tal12a-dependent susceptibility. COMPETING INTEREST Authors declare no competing interests AUTHORS CONTRIBUTIONS CA, LDN, IF and CB performed preliminary proof-of-concept tests. BC, ZED, AJB, LDN and CA participated in the experimental design and conception of the study. BC carried out all the experiments, performed the statistical analysis, BC and CA wrote the manuscript. CG and BC cloned the Tal genes and introduced them in the effectorless Xanthomonas strain. CB and IF isolated the CN08 mutant and complemented the strain. SC and BC analyzed and interpreted the transcriptomic data. ALPQ predicted the EBE. ALR performed the imageJ analysis. All co-authors reviewed and corrected the manuscript. DATA AVAILABILITY Raw sequence data for the transcriptomic analysis are available on the Sequence Read Archive (SRA) database under accession number SRP523615. FUNDING INFORMATIONS The authors are grateful to the funding sources: B.C. was funded by a PhD grant from the INRAE BAP division. C.G., S.C., C.B., I.F. and L.D.N. were supported by the NEPHRON ANR project (ANR-18-CE20-0020-01). This study is set within the framework of the ‘Laboratoires d’Excellences’ (LABEX) TULIP (ANR-10-LABX-41) and of the ‘Ecole Universitaire de Recherche’ (EUR) TULIP-GS (ANR-18-EURE-0019). Z.D. was supported by a USDA NIFA Pre-doctoral Research Fellowship (2019-67011-29501). ACKNOWLEDGEMENTS We are grateful to Boris Szurek (PHIM, Montpellier, France) for advice on the design and execution of the study. Funder Information Declared NEPHRON ANR , ANR-18-CE20-0020-01 LABEX TULIP , ANR-10-LABX-41 EUR TULIP-GS , ANR-18-EURE-0019 USDA NIFA Pre-doctoral Research Fellowship , 2019-67011-29501 INRAE BAP REFERENCES ↵ Aboyoun P , Pages H , Lawrence M . 2013 . GenomicRanges: representation and manipulation of genomic intervals . R package version 1 . 12 . 5 . OpenUrl ↵ Alexa A , Rahnenführer J , Lengauer T . 2006 . Improved scoring of functional groups from gene expression data by decorrelating GO graph structure . Bioinformatics 22 : 1600 – 1607 . OpenUrl CrossRef PubMed Web of Science ↵ An S-Q , Potnis N , Dow M , Vorhölter F-J , He Y-Q , Becker A , Teper D , Li Y , Wang N , Bleris L , et al. 2020 . Mechanistic insights into host adaptation, virulence and epidemiology of the phytopathogen Xanthomonas . FEMS Microbiology Reviews 44 : 1 – 32 . 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Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following A Xanthomonas effector protein contributes quantitatively to virulence by inducing at least two minor Susceptibility genes Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share A Xanthomonas effector protein contributes quantitatively to virulence by inducing at least two minor Susceptibility genes Brice Charleux , Carine Gris , Sébastien Carrère , Alvaro L. Pérez-Quintero , Aurélie Le Ru , Caroline Bellenot , Ivanna Fuentes , Zoë E. Dubrow , Adam J. Bogdanove , Laurent D. 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