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The bacterial pathogens Xanthomonas oryzae pathovars oryzae (Xoo) and oryzicola (Xoc) cause leaf blight and leaf streak diseases on rice, respectively. Pathogenesis is largely defined by the virulence genes harboured in the pathogen genome. Recently, we demonstrated that the protein HpaP of the crucifer pathogen Xanthomonas campestris pv. campestris is an enzyme with both ATPase and phosphatase activities, and is involved in regulating the synthesis of virulence factors and the induction of the hypersensitive response (HR). In this study, we investigated the role of HpaP homologues in Xoo and Xoc. We showed that HpaP is required for full virulence of Xoo and Xoc. Deletion of hpaP in Xoo and Xoc led to a reduction in virulence and alteration in the production of virulence factors, including extracellular polysaccharide and cell motility. Comparative transcriptomics and reverse transcription-quantitative PCR assays revealed that in XVM2 medium, a mimic medium of the plant environment, the expression levels of hrp genes (for HR and pathogenicity) were enhanced in the Xoo hpaP deletion mutant compared to the wild type. By contrast, in the same growth conditions, hrp gene expression was decreased in the Xoc hpaP deletion mutant compared to the wild type. However, an opposite expression pattern was observed when the pathogens grew in planta, where the expression of hrp genes was reduced in the Xoo hpaP mutant but increased in the Xoc hpaP mutant. These findings indicate that HpaP plays a divergent role in Xoo and Xoc, which may lead to the different infection strategies employed by these two pathogens.

Type 6 secretion systems (T6SSs) are specialized multiprotein complexes that inject protein effectors into prokaryotic and/or eukaryotic cells. We previously described the role of the T6SS of the phytopathogen Xanthomonas citri pv. citri as an anti-eukaryotic nanoweapon that confers resistance to predation by the amoeba Dictyostelium discoideum. Transcription of the X. citri T6SS genes is induced by a signaling cascade involving the Ser/Thr kinase PknS and the extracytoplasmic function sigma factor EcfK. Here, we used a strain overexpressing a phosphomimetic constitutively active version of EcfK (EcfK

The Xanthomonas transcription activator-like effector (TALE) protein AvrBs3 transcriptionally activates the executor-type resistance (R) gene Bs3 from pepper (Capsicum annuum), thereby triggering a hypersensitive cell death reaction (HR). AvrBs3 also triggers an HR in tomato (Solanum lycopersicum) upon recognition by the nucleotide-binding leucine-rich repeat (NLR) R protein Bs4. Whether the executor-type R protein Bs3 and the NLR-type R protein Bs4 use common or distinct signalling components to trigger an HR remains unclear. CRISPR/Cas9-mutagenesis revealed, that the immune signalling node EDS1 is required for Bs4- but not for Bs3-dependent HR, suggesting that NLR- and executor-type R proteins trigger an HR via distinct signalling pathways. CRISPR/Cas9-mutagenesis also revealed that tomato Bs4 suppresses the virulence function of both TALEs, the HR-inducing AvrBs3 protein and of AvrHah1, a TALE that does not trigger an HR in tomato. Analysis of AvrBs3- and AvrHah1-induced host transcripts and disease phenotypes in CRISPR/Cas9-induced bs4 mutant plants indicates that both TALEs target orthologous transcription factor genes to promote disease in tomato and pepper host plants. Our studies display that tomato mutants lacking the TALE-sensing Bs4 protein provide a novel platform to either uncover TALE-induced disease phenotypes or genetically dissect components of executor-triggered HR.

Plant secondary metabolites perform numerous functions in the interactions between plants and pathogens. However, little is known about the precise mechanisms underlying their contribution to the direct inhibition of pathogen growth and virulence in planta. Here, we show that the secondary metabolite sulforaphane (SFN) in crucifers inhibits the growth, virulence, and ability of Xanthomonas species to adapt to oxidative stress, which is essential for the successful infection of host plants by phytopathogens. The transcription of oxidative stress detoxification-related genes (catalase [katA and katG] and alkylhydroperoxide-NADPH oxidoreductase subunit C [ahpC]) was substantially inhibited by SFN in Xanthomonas campestris pv. campestris (Xcc), and this phenomenon was most obvious in sax gene mutants sensitive to SFN. By performing microscale thermophoresis (MST) and electrophoretic mobility shift assay (EMSA), we observed that SFN directly bound to the virulence-related redox-sensing transcription factor OxyR and weakened the ability of OxyR to bind to the promoters of oxidative stress detoxification-related genes. Collectively, these results illustrate that SFN directly targets OxyR to inhibit the bacterial adaptation to oxidative stress, thereby decreasing bacterial virulence. Interestingly, this phenomenon occurs in multiple Xanthomonas species. This study provides novel insights into the molecular mechanisms by which SFN limits Xanthomonas adaptation to oxidative stress and virulence, and the findings will facilitate future studies on the use of SFN as a biopesticide to control Xanthomonas.

Gradually aggravated disease caused by phytopathogenic bacteria severely restricts food security and crop yield, and few pesticides can relieve this severe situation. Thus, development and excavation of new agrochemicals with high bioactivity and novel action mechanism may be a feasible strategy to control intractable bacterial diseases. As a privileged molecular framework, steroid molecules exhibit diversiform bioactivities. Herein, a series of novel androst-4-ene derivatives were designed, synthesised and investigated for their antibacterial behaviour to excavate novel agrochemicals on the base of steroid molecules.Bioassay results indicated that target compounds displayed high bioactivities toward three destructive phytopathogenic bacteria, including Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas axonopodis pv. citri (Xac) and Pseudomonas syringae pv. actinidiae (Psa). Compound IIIGiven these profiles of target compounds, we highlight a new strategy for controlling intractable plant bacterial diseases by inducing plant resistance and targeting the bacterial cell membrane. © 2022 Society of Chemical Industry.

Lack of control agents and development of bacterial resistance are emergent problems in the chemical control of rice bacterial blight, therefore novel bactericides against Xanthomonas oryzae pv. oryzae (Xoo, the causal agent of rice bacterial blight) are urgently needed. We previously found that parthenolide (PTL) is a potential lead against Xoo, and PTL inhibits Xoo growth via oxidative stress. However, the mechanism of action of PTL against Xoo needs further elucidation.In this study, a biotinylated PTL probe was synthesized, and two important subunits in the respiratory chain (NuoF of complex I and SdhB of complex II) of Xoo were captured with the probe and identified with liquid chromatography tandem mass spectrometry (LC-MS/MS). The binding between them was verified with pull-down and drug affinity responsive target stability technologies. In addition, purified proteins of NuoF and SdhB greatly lowered the antibacterial activity of PTL, and PTL evidently inhibited the enzyme activities of complexes I and II. Moreover, knockout of nuoF and sdhB in Xoo caused elevated reactive oxygen species (ROS) levels and increased sensitivity to PTL. Furthermore, molecular simulations indicated that PTL may form covalent bonds with Cys105 and Cys187 in NuoF and Cys106 in SdhB.PTL can directly bind to NuoF and SdhB, which impairs the enzyme functions of complexes I and II in the respiratory chain, leading to ROS accumulation in Xoo. This study will provide deep insight into the mechanism of action of PTL against Xoo. © 2022 Society of Chemical Industry.

Cellobiosidase (CbsA) is an important secreted virulence factor of Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight of rice. CbsA is one of several cell wall-degrading enzymes secreted by Xoo via the type II secretion system (T2SS). CbsA is considered a fundamental virulence factor for vascular pathogenesis. CbsA has an N-terminal glycosyl hydrolase domain and a C-terminal fibronectin type III (FnIII) domain. Interestingly, the secreted form of CbsA lacks the FnIII domain during in planta growth. Here we show that the presence of the FnIII domain inhibits the enzyme activity of CbsA on polysaccharide substrates like carboxymethylcellulose. The FnIII domain is required for the interaction of CbsA with SecB chaperone, and this interaction is crucial for the stability and efficient transport of CbsA across the inner membrane. Deletion of the FnIII domain reduced virulence similar to ΔcbsA Xoo, which corroborates the importance of the FnIII domain in CbsA. Our work elucidates a hitherto unknown function of the FnIII domain in enabling the virulence-promoting activity of CbsA.

SWEETs play important roles in intercellular sugar transport. Induction of SWEET sugar transporters by Transcription Activator-Like effectors (TALe) of Xanthomonas ssp. is key for virulence in rice, cassava and cotton. We identified OsSWEET11b with roles in male fertility and potential bacterial blight (BB) susceptibility in rice. While single ossweet11a or 11b mutants were fertile, double mutants were sterile. As clade III SWEETs can transport gibberellin (GA), a key hormone for spikelet fertility, sterility and BB susceptibility might be explained by GA transport deficiencies. However, in contrast with the Arabidopsis homologues, OsSWEET11b did not mediate detectable GA transport. Fertility and susceptibility therefore are likely to depend on sucrose transport activity. Ectopic induction of OsSWEET11b by designer TALe enabled TALe-free Xanthomonas oryzae pv. oryzae (Xoo) to cause disease, identifying OsSWEET11b as a potential BB susceptibility gene and demonstrating that the induction of host sucrose uniporter activity is key to virulence of Xoo. Notably, only three of six clade III SWEETs are targeted by known Xoo strains from Asia and Africa. The identification of OsSWEET11b is relevant for fertility and for protecting rice against emerging Xoo strains that target OsSWEET11b.

The rice immune receptor XA21 confers resistance to Xanthomonas oryzae pv. oryzae (Xoo), and upon recognition of the RaxX21-sY peptide produced by Xoo, XA21 activates the plant immune response. Here we screened 21 000 mutant plants expressing XA21 to identify components involved in this response, and reported here the identification of a rice mutant, sxi4, which is susceptible to Xoo. The sxi4 mutant carries a 32-kb translocation from chromosome 3 onto chromosome 7 and displays an elevated level of DCL2a transcript, encoding a Dicer-like protein. Silencing of DCL2a in the sxi4 genetic background restores resistance to Xoo. RaxX21-sY peptide-treated leaves of sxi4 retain the hallmarks of XA21-mediated immune response. However, WRKY45-1, a known negative regulator of rice resistance to Xoo, is induced in the sxi4 mutant in response to RaxX21-sY peptide treatment. A CRISPR knockout of a short interfering RNA (TE-siRNA815) in the intron of WRKY45-1 restores the resistance phenotype in sxi4. These results suggest a model where DCL2a accumulation negatively regulates XA21-mediated immunity by altering the processing of TE-siRNA815.

To better explore the potential of rice extra-large G (XLG) proteins in future breeding, we characterised the function of OsXLG1, OsXLG2 and OsXLG3 in disease resistance. Loss-of-function Osxlg2 and Osxlg3 mutants showed reduced resistance to the fungal pathogen Magnaporthe oryzae, whereas Osxlg1 mutants were specifically compromised in resistance to the bacterial pathogen Xanthomonas oryzae pv oryzae. Consistent with their effects on rice blast resistance, mutations in OsXLG2 and OsXLG3 caused greater defects than did mutations in OsXLG1 for chitin-induced defence responses. All three OsXLGs interacted with components of a surface immune receptor complex composed of OsCERK1, OsRLCK176 and OsRLCK185. Further characterisation of yield-related traits showed that the Osxlg3 mutants displayed reduced plant height, panicle length and 1000grain weight, whereas Osxlg1 mutants exhibited increased plant height, panicle length and 1000-grain weight. Together the study shows the differential contributions of the three OsXLG proteins to disease resistance to fungal and bacterial pathogens, their yield-related traits and provides insights for future improvement of rice production.

Bacterial blight (BB) of rice is a devastating disease caused by Xanthomonas oryzae pv. oryzae (Xoo). The evolution of new pathogenic races of bacterial blight pathogen is always a potential threat for rice production. The deployment of pathotype-specific resistant genes in the host plants is a feasible strategy to develop BB-resistant varieties. Therefore, continuous disease monitoring, identification of Xoo pathotypes, and their distribution are crucial to managing BB. In this study, 71 Xoo isolates were collected from the Godavari delta in Andhra Pradesh (India) and their virulence profiles on rice BB differentials were characterized. Data revealed that different International Rice Bacterial Blight (IRBB) lines with single BB resistance genes were susceptible to 73.2%-97.2% of the isolates, except IRBB13 (possessing BB resistance gene, xa13) which showed a moderately susceptible or susceptible reaction to 47.9% of the isolates. Three gene combination rice differentials like IRBB56 (Xa4 + xa5 + xa13), IRBB57 (Xa4 + xa5 + Xa21), IRBB58 (Xa4 + xa13 + Xa21), and IRBB59 (xa5 + xa13 + Xa21) showed very broad-spectrum resistance to majority of the Xoo isolates from the region. None of the tested Xoo isolates were virulent on IRBB58 (Xa4 + xa13 + Xa21), IRBB60 (Xa4 + xa5 + xa13 + Xa21), and IRBB66 (Xa4 + xa5 + Xa7 + xa13 + Xa21). Based on the virulence reaction, 71 Xoo isolates were grouped into 10 major pathotypes. Highly virulent pathotypes viz., IXoPt # 14, 17, 19, and 22 can break the resistance of major BB-resistant genes and were commonly distributed throughout the surveyed regions. Genotypic data of 71 Xoo isolates using J3 primer divided them into three major clusters. Cluster I consisted of 24 Xoo isolates that belonged to pathotype IXoPt-19. Cluster II consisted of 41 Xoo isolates belonging to seven different pathotypes, and Cluster III was composed of six isolates from three different pathotypes. The findings of this study will be helpful to develop rice varieties with pathotype-specific broad-spectrum resistance against BB.

Natural products are a major source of new antibiotics. Here we utilize biosynthetic instructions contained within metagenome-derived congener biosynthetic gene clusters (BGCs) to guide the synthesis of improved antibiotic analogues. Albicidin and cystobactamid are the first members of a new class of broad-spectrum ρ-aminobenzoic acid (PABA)-based antibiotics. Our search for PABA-specific adenylation domain sequences in soil metagenomes revealed that BGCs in this family are common in nature. Twelve BGCs that were bio-informatically predicted to encode six new congeners were recovered from soil metagenomic libraries. Synthesis of these six predicted structures led to the identification of potent antibiotics with changes in their spectrum of activity and the ability to circumvent resistance conferred by endopeptidase cleavage enzymes.

Xanthomonas oryzae pv. oryzae (Xoo) causes a serious disease of rice known as bacterial leaf blight. Several virulence-associated functions have been characterized in Xoo. However, the role of important second messenger c-di-GMP signalling in the regulation of virulence-associated functions still remains elusive in this phytopathogen. In this study we have performed an investigation of 13 c-di-GMP modulating deletion mutants to understand their contribution in Xoo virulence and lifestyle transition. We show that four Xoo proteins, Xoo2331, Xoo2563, Xoo2860 and Xoo2616, are involved in fine-tuning the in vivo c-di-GMP abundance and also play a role in the regulation of virulence-associated functions. We have further established the importance of the GGDEF domain of Xoo2563, a previously characterized c-di-GMP phosphodiesterase, in the virulence-associated functions of Xoo. Interestingly the strain harbouring the GGDEF domain deletion (ΔXoo2563GGDEF ) exhibited EPS deficiency and hypersensitivity to streptonigrin, indicative of altered iron metabolism. This is in contrast to the phenotype exhibited by an EAL overexpression strain wherein, the ΔXoo2563GGDEF exhibited other phenotypes, similar to the strain overexpressing the EAL domain. Taken together, our results indicate a complex interplay of c-di-GMP signalling with the cell-cell signalling to coordinate virulence-associated function in Xoo.

The natural product albicidin is a highly potent inhibitor of bacterial DNA gyrase. Its outstanding activity, particularly against Gram‐negative pathogens, qualifies it as a promising lead structure in the search for new antibacterial drugs. However, as we show here, the N‐terminal cinnamoyl moiety of albicidin is susceptible to photochemical E/Z isomerization. Moreover, the newly formed Z isomer exhibits significantly reduced antibacterial activity, which hampers the development and biological evaluation of albicidin and potent derivatives thereof. Hence, we synthesized 13 different variants of albicidin in which the vulnerable para‐coumaric acid moiety was replaced; this yielded photostable analogues. Biological activity assays revealed that diaryl alkyne analogues exhibited virtually undiminished antibacterial efficacy. This promising scaffold will therefore serve as a blueprint for the design of a potent albicidin‐based drug., Lead optimization: The cinnamoyl residue of the antibiotic albicidin is shown to readily undergo UV‐mediated E/Z isomerization, thereby converting from the highly bioactive E form found in the natural product to the significantly less bioactive Z form. Acetylenic replacement of the susceptible methylacrylamide moiety affords a set of photochemically stable albicidin derivatives with virtually undiminished antibacterial activity.

Summary RNase P functions either as a catalytic ribonucleoprotein (RNP) or as an RNA‐free polypeptide to catalyse RNA processing, primarily tRNA 5′ maturation. To the growing evidence of non‐canonical roles for RNase P RNP subunits including regulation of chromatin structure and function, we add here a role for the rice RNase P Rpp30 in innate immunity. This protein (encoded by LOC_Os11g01074) was uncovered as the top hit in yeast two‐hybrid assays performed with the rice histone deacetylase HDT701 as bait. We showed that HDT701 and OsRpp30 are localized to the rice nucleus, OsRpp30 expression increased post‐infection by Pyricularia oryzae (syn. Magnaporthe oryzae), and OsRpp30 deacetylation coincided with HDT701 overexpression in vivo. Overexpression of OsRpp30 in transgenic rice increased expression of defence genes and generation of reactive oxygen species after pathogen‐associated molecular pattern elicitor treatment, outcomes that culminated in resistance to a fungal (P. oryzae) and a bacterial (Xanthomonas oryzae pv. oryzae) pathogen. Knockout of OsRpp30 yielded the opposite phenotypes. Moreover, HA‐tagged OsRpp30 co‐purified with RNase P pre‐tRNA cleavage activity. Interestingly, OsRpp30 is conserved in grass crops, including a near‐identical C‐terminal tail that is essential for HDT701 binding and defence regulation. Overall, our results suggest that OsRpp30 plays an important role in rice immune response to pathogens and provides a new approach to generate broad‐spectrum disease‐resistant rice cultivars.

A few reports have indicated that a single gene confers resistance to bacterial blight, sheath blight and rice blast. In this study, we identified a novel disease resistance mutant gene, methyl esterase‐like (osmesl) in rice. Mutant rice with T‐DNA insertion displayed significant resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo), sheath blight caused by Rhizoctonia solani and rice blast caused by Magnaporthe oryzae. Additionally, CRISPR‐Cas9 knockout mutants and RNAi lines displayed resistance to these pathogens. Complementary T‐DNA mutants demonstrated a phenotype similar to the wild type (WT), thereby indicating that osmesl confers resistance to pathogens. Protein interaction experiments revealed that OsMESL affects reactive oxygen species (ROS) accumulation by interacting with thioredoxin OsTrxm in rice. Moreover, qRT‐PCR results showed significantly reduced mRNA levels of multiple ROS scavenging‐related genes in osmesl mutants. Nitroblue tetrazolium staining showed that the pathogens cause ROS accumulation, and quantitative detection revealed significantly increased levels of H2O2 in the leaves of osmesl mutants and RNAi lines after infection. The abundance of JA, a hormone associated with disease resistance, was significantly more in osmesl mutants than in WT plants. Overall, these results suggested that osmesl enhances disease resistance to Xoo, R. solani and M. oryzae by modulating the ROS balance.

Xanthomonas albilineans (Xa) and X. sacchari (Xs) are both sugarcane pathogens. Xa is the causal agent of leaf scald disease, but there is limited information about the pathogenicity of Xs. The aim of this work was to study virulence factors of native strains of Xa (Xa32, Xa33, and XaM6) and Xs (Xs14 and Xs15) previously isolated from sugarcane with leaf scald symptoms, to gain insight into the biology of each microorganism. We analysed epiphytic survival, sensitivity to oxidative stress, extracellular degradative enzymes, cell motilities, exopolysaccharide (EPS) characteristics, cell adhesion, biofilm development, and control of stomatal regulation of the five strains. We observed that each species presented similar phenotypes for every factor analysed. Xa strains appeared to be more sensitive to oxidative stress and presented lower epiphytic survival than Xs. All strains presented endoglucanase activity; however, we could only detect protease and amylase activities in Xs strains. Swimming and sliding were higher in Xs, but twitching was variable among species. We also observed that only Xs strains produced a xanthan-like EPS, presented a strong cell adhesion, and structured biofilm. We detected some intraspecific variations showing that higher amounts of EPS produced by Xs14 correlated with its higher sliding motility and its homogenous and more adhesive biofilm. In addition, EPSs of Xs14 and Xs15 presented differences in strand height and acetyl percentage. Finally, we found that strains of both species were able to interfere with stomatal aperture mechanism. All these differences could influence the colonization strategies and/or disease progression in each species. Fil: Mielnichuk, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina Fil: Bianco, María Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina Fil: Yaryura, Pablo Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones y Transferencia de Villa María. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; Argentina Fil: Bertani, Romina Priscila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnología Agroindustrial del Noroeste Argentino; Argentina Fil: Toum, Laila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina Fil: Daglio, Yasmin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Colonnella, Maria Antonela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina Fil: Lizarraga, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina Fil: Castagnaro, Atilio Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnología Agroindustrial del Noroeste Argentino; Argentina Fil: Vojnov, Adrián Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina

Citrus sinensis lateral organ boundary 1 (CsLOB1) was previously identified as a critical disease susceptibility gene for citrus bacterial canker, which is caused by Xanthomonas citri subsp. citri (Xcc). However, the molecular mechanisms of CsLOB1 in citrus response to Xcc are still elusive. Here, we constructed transgenic plants overexpressing and RNAi-silencing of CsLOB1 using the canker-disease susceptible 'wanjincheng' orange (C. sinensis Osbeck) as explants. CsLOB1-overexpressing plants exhibited dwarf phenotypes with smaller and thicker leaf, increased branches and adventitious buds clustered on stems. These phenotypes were followed by a process of pustule- and canker-like development that exhibited enhanced cell proliferation. Pectin depolymerization and expansin accumulation were enhanced by CsLOB1 overexpression, while cellulose and hemicellulose synthesis were increased by CsLOB1 silence. Whilst overexpression of CsLOB1 increased susceptibility, RNAi-silencing of CsLOB1 enhanced resistance to canker disease without impairing pathogen entry. Transcriptome analysis revealed that CsLOB1 positively regulated cell wall degradation and modification processes, cytokinin metabolism, and cell division. Additionally, 565 CsLOB1-targeted genes were identified in chromatin immunoprecipitation-sequencing (ChIP-seq) experiments. Motif discovery analysis revealed that the most highly overrepresented binding sites had a conserved 6-bp 'GCGGCG' consensus DNA motif. RNA-seq and ChIP-seq data suggested that CsLOB1 directly activates the expression of four genes involved in cell wall remodeling, and three genes that participate in cytokinin and brassinosteroid hormone pathways. Our findings indicate that CsLOB1 promotes cell proliferation by mechanisms depending on cell wall remodeling and phytohormone signaling, which may be critical to citrus canker development and bacterial growth in citrus.

Transcription activator-like effectors (TALEs), which induce the expression of specific plant genes to promote infection, are the main pathogenic determinants of various Xanthomonas bacteria. However, investigation of TALEs from Xanthomonas campestris pv. campestris, which causes black rot disease of crucifers, received little attention. In this study, we used PCR-based amplification followed by SMRT amplicon sequencing to identify TALE genes in several X. campestris pv. campestris strains. Computational prediction in conjunction with quantitative reverse transcription PCR analysis was used to find their targets in the Brassica oleracea genome. Transcription factor ERF121, from the AP2/ERF family, was identified as target gene for the conserved TALEs from multiple X. campestris pv. campestris strains. Several members of this family from diverse plants were previously identified as targets of TALEs from different Xanthomonas species. We propose that TALE-dependent activation of AP2/ERF transcription factors promotes susceptibility to Xanthomonas through the misregulation of plant defence pathways.

Bacterial spot, which is caused by several Xanthomonas species, is an economically important disease in tomato (Solanum lycopersicum). Great efforts have been made for the identification of resistant sources and the genetic analysis of resistance. However, the development of resistant commercial varieties is slow due to the existence of multiple species of the pathogen and a poor understanding of the resistance mechanism in tomato. The current study revealed that the Rx4 gene encodes a nucleotide-binding leucine-rich repeat protein in the wild tomato species Solanum pimpinellifolium and specifically recognizes and confers a hypersensitive response (HR) to Xanthomonas euvesicatoria pv. perforans race T3 expressing the AvrXv3 avirulence protein. Complementation of the Rx4 gene in the susceptible tomato line Ohio 88119 using a transgenic approach resulted in HR, whereas knockout of the gene through CRISPR/Cas9 editing in resistant lines Hawaii 7981 and PI 128216 led to non-HR to race T3. Transcription of Rx4 was not induced by the presence of race T3. Furthermore, the Rx4 protein did not show physical interaction with AvrXv3 but interacted with SGT1-1 and RAR1. Virus-induced gene silencing of SGT1-1 and RAR1 in the resistant line PI128216 suppressed the HR to race T3. Taken together, our study confirms Rx4 is the gene conferring the HR to bacterial spot race T3 and reveals the potential roles of SGT1-1 and RAR1 as signals in the Rx4-mediated HR. This discovery represents a step forward in our understanding of the mechanism of resistance to bacterial spot in tomato and may have important implications for understanding plant-bacterial interactions.

BACKGROUND Xanthomonas axonopodis pv. glycines (Xag) is a hazardous pathogen able to cause bacterial pustule disease in soybean, reducing crop yield and quality. Although flavonoids rutin and genistein are known to play an important role in soybean defence, soybean is only able to produce Biochanin A in low concentration. RESULTS In this work, Biochanin A was found to produce higher antibacterial activity against Xag in comparison with genistein (minimum inhibitory concentration

For decades, Xanthomonas vasicola pv. musacearum (Xvm) has been an economically important bacterial pathogen on enset in Ethiopia. Since 2001, Xvm has also been responsible for significant losses to banana crops in several East and Central African countries, with devastating consequences for smallholder farmers. Understanding the genetic diversity within Xvm populations is essential for the smart design of transnationally reasoned, durable, and effective management practices. Previous studies have revealed limited genetic diversity in Xvm, with East African isolates from banana each falling into one of two closely related clades previously designated as sublineages SL 1 and SL 2, the former of which had also been detected on banana and enset in Ethiopia. Given the presumed origin of Xvm in Ethiopia, we hypothesized that both clades might be found in that country, along with additional genotypes not seen in Central and East African bananas. Genotyping of 97 isolates and whole‐genome sequencing of 15 isolates revealed not only the presence of SL 2 in Ethiopia, but additional diversity beyond SL 1 and SL 2 in four new clades. Moreover, SL 2 was detected in the Democratic Republic of Congo, where previously SL 1 was the only clade reported. These results demonstrate a greater range of genetic diversity among Xvm isolates than previously reported, especially in Ethiopia, and further support the hypothesis that the East/Central Africa xanthomonas wilt epidemic has been caused by a restricted set of genotypes drawn from a highly diverse pathogen pool in Ethiopia.\ud \ud

Made available in DSpace on 2021-06-25T10:37:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-04-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) BACKGROUND: The largest and most profitable market for citrus is the production of fresh fruit. Xanthomonas citri subsp. citri is a Gram-negative plant pathogen and the etiological agent of citrus canker, one of the major threats to citrus production worldwide. In the early stages of infection, X. citri can attach to plant surfaces by means of biofilms. Biofilm is considered an essential virulence factor, which helps tissue colonization in plants. Thus, sanitization of citrus fruit is mandatory in packinghouses before any logistic operation as packing and shipment to the market. The aim of this study was to evaluate electrolysed water (EW) as a sanitizer for the disinfection of citrus fruit in packinghouses. RESULTS: Using a protocol to monitor cell respiration we show that EW, obtained after 8 and 9 min of electrolysis, sufficed to kill X. citri when applied at a concentration of 500 μL mL−1. Furthermore, microscopy analysis, combined with time-response growth curves, confirmed that EW affects the bacterial cytoplasmatic membrane and it leads to cell death in the first few minutes of contact. Pathogenicity tests using limes to simulate packinghouse treatment showed that EW, produced with 9 min of electrolysis, was a very effective sanitizer capable of eliminating X. citri from contaminated fruit. CONCLUSION: It was possible to conclude that EW is significantly effective as sodium hypochlorite (NaClO) at 200 ppm. Therefore, EW could be an alternative for citrus sanitization in packinghouses. © 2020 Society of Chemical Industry. Department of Biochemistry and Microbiology Sao Paulo State University (UNESP) Department of Natural Sciences Mathematics and Education Agricultural Sciences Centre Federal University of Sao Carlos (UFSCar) Department of Biochemistry and Microbiology Sao Paulo State University (UNESP) FAPESP: 2017/07306-9

Background The emergence of drug-resistant phytopathogenic bacteria and the need for new type of biological disease-control agents have accelerated efforts toward searching for alternative candidates with a low propensity for resistance development. In this study, a new series of stilbene-based peptoid mimics were synthesized, and their biological activities were evaluated against citrus pathogenic bacteria in vitro and in vivo. Results Antibacterial bioassay results showed that the dicationic peptoid mimics 9a and 9b displayed excellent bioactivity against Xanthomonas axonopodis pv citri, with the minimum inhibitory concentration (MIC) values of 25 μM, which were superior to those of commercial copper biocides Delite (200 μM) and Kasumin Bordeaux (100 μM). In vivo bioassay further confirmed their control efficacy against plant bacterial diseases. In addition, the antibacterial mechanism of action elucidated their membrane-disruption effects resulting in the leakage of the bacterial membranes, which was similar to that of antimicrobial peptides (AMPs). Moreover, the inhibition effect on biofilm formation of peptoid mimics has also been demonstrated. Conclusion Stilbene-based peptoid mimics synthesized in this study showed promising antibacterial activity with a potent membrane-disruptive mechanism. The results suggested that stilbene-based peptoid mimics have the potential as a new type of bactericide candidates for citrus disease protection. This article is protected by copyright. All rights reserved.

Background The rice bacterial leaf blight (BLB) is one of the most serious bacterial diseases caused by Xanthomonas oryzae pv. oryzae (Xoo), which can cause yield loss of rice up to 50%. The three-dimensional quantitative structure-activity relationship (3D-QSAR) is an important auxiliary method to find potential high-efficient pesticides active structures. Results A series of novel 1,3,4-oxadiazole compounds were designed and synthesized based on the 3D-QSAR model, and their antibacterial activities in vitro against Xoo were evaluated. The results indicated that all the target compounds showed excellent in vitro antibacterial activities. For example, the compounds 6, 12, 13, 20, 21, and 23 exhibited excellent antibacterial activities against Xoo, with half-maximal effective concentration (EC50 ) values of 0.24, 0.31, 0.36, 0.29, 0.19, and 0.31 mg/L, respectively, which were superior to the antibacterial agents thiodiazole copper (127.44 mg/L) and bismerthiazol (91.08 mg/L). Meanwhile, compound 21 showed good antibacterial activity in vivo against BLB, with curative and protective activities of 46.7% and 56.4%, respectively, which were superior to thiodiazole copper (28.5% and 32.5%) and bismerthiazol (37.6% and 38.4%). Compound 21 can significantly reduce the extracellular polysaccharides production of Xoo, increase the permeability of the cell membranes, and also can cause cell surface wrinkles, deformation and dryness. Conclusion The 3D-QSAR model can be used to find sulfone compounds containing a 1,3,4-oxadiazole moiety with higher antibacterial activity, and compound 21 can be used as a potential antibacterial agent in the future. © 2020 Society of Chemical Industry.

Xanthomonas oryzae pv. oryzae (Xoo) is a vascular pathogen that causes leaf blight in rice, leading to severe yield losses. Since the usage of chemical control methods has not been very promising for the future disease management, it is of high importance to systematically gain new insights about Xoo virulence and pathogenesis, and devise effective strategies to combat the rice disease. To do this, we reconstructed a genome‐scale metabolic model of Xoo (iXOO673) and validated the model predictions using culture experiments. Comparison of the metabolic architecture of Xoo and other plant pathogens indicated that the Entner–Doudoroff pathway is a more common feature in these bacteria than previously thought, while suggesting some of the unique virulence mechanisms related to Xoo metabolism. Subsequent constraint‐based flux analysis allowed us to show that Xoo modulates fluxes through gluconeogenesis, glycogen biosynthesis, and degradation pathways, thereby exacerbating the leaf blight in rice exposed to nitrogenous fertilizers, which is remarkably consistent with published experimental literature. Moreover, model‐based interrogation of transcriptomic data revealed the metabolic components under the diffusible signal factor regulon that are crucial for virulence and survival in Xoo. Finally, we identified promising antibacterial targets for the control of leaf blight in rice by using gene essentiality analysis., We reconstructed a genome‐scale metabolic model of the rice leaf pathogen Xanthomonas oryzae for understanding its virulence and survival, and identified promising antibacterial targets.

Xanthomonas campestris pv. campestris (Xcc) controls virulence and plant infection mechanisms via the activity of the sensor kinase and response regulator pair HpaS/hypersensitive response and pathogenicity G (HrpG). Detailed analysis of the regulatory role of HpaS has suggested the occurrence of further regulators besides HrpG. Here we used in vitro and in vivo approaches to identify the orphan response regulator VemR as another partner of HpaS and to characterize relevant interactions between components of this signalling system. Bacterial two‐hybrid and protein pull‐down assays revealed that HpaS physically interacts with VemR. Phos‐tag SDS‐PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of VemR in vivo. Mutation analysis reveals that HpaS and VemR contribute to the regulation of motility and this relationship appears to be epistatic. Additionally, we show that VemR control of Xcc motility is due in part to its ability to interact and bind to the flagellum rotor protein FliM. Taken together, the findings describe the unrecognized regulatory role of sensor kinase HpaS and orphan response regulator VemR in the control of motility in Xcc and contribute to the understanding of the complex regulatory mechanisms used by Xcc during plant infection., Sensor kinase HpaS regulates T3SS and swimming motility by controlling the phosphorylation of response regulators HrpG and VemR, respectively.

International audience; At the colonization site of a foreign entity, plant cells alter their trajectory of growth and development. The resulting structure – a plant gall – accommodates various needs of the foreigner, which are phylogenetically diverse: viruses, bacteria, protozoa, oomycetes, true fungi, parasitic plants, and many types of animals, including rotifers, nematodes, insects, and mites. The plant species that make galls also are diverse. We assume gall production costs the plant. All is well if the foreigner provides a gift that makes up for the cost. Nitrogen‐fixing nodule‐inducing bacteria provide nutritional services. Gall wasps pollinate fig trees. Unfortunately for plants, most galls are made for foes, some of which are deeply studied pathogens and pests: Agrobacterium tumefaciens, Rhodococcus fascians, Xanthomonas citri, Pseudomonas savastanoi, Pantoea agglomerans, ‘Candidatus’ phytoplasma, rust fungi, Ustilago smuts, root knot and cyst nematodes, and gall midges. Galls are an understudied phenomenon in plant developmental biology. We propose gall inception for discovering unifying features of the galls that plants make for friends and foes, talk about molecules that plants and gall‐inducers use to get what they want from each other, raise the question of whether plants colonized by arbuscular mycorrhizal fungi respond in a gall‐like manner, and present a research agenda.

Pomelo seeds (PS) are important by-product of pomelo fruits (Citrus grandis Osbeck). The value-added utilization of PS remains highly challenged. This study aimed to investigate the utilization potential of PS as natural antioxidant, antibacterial, herbicidal agents, and their functional components. The ethanolic extract (EE) of PS and its four fractions as PEE (petroleum ether extract), AcOEtE (ethyl acetate extract), BTE (butanol extract), and WE (water extract), were prepared and biologically evaluated. BTE exhibited the best antioxidant activity among all these extracts, in both ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) and FRAP (ferric reducing antioxidant power) assays. AcOEtE was superior to other extracts in herbicidal assay against both Festuca elata Keng (IC50 of 0.48 mg mL-1 ) and Amaranthus retroflexus L. (IC50 of 0.94 mg mL-1 ). Meanwhile, both AcOEtE and BTE demonstrated inhibitory effects against Bacillus subtilis, Escherichia coli, and Xanthomonas citri subsp. citri, with MIC ranging 2.5-5.0 mg mL-1 . Furthermore, the primary chemical components involving naringin, deacetylnomilin, limonin, nomilin, and obacunone, were quantified in all these extracts. PCA (principal component analysis) suggested that naringin might highly contribute to the antioxidant activity of PS, and the herbicidal activity should be ascribed to limonoids. This study successfully identified AcOEtE and BTE as naturally occurring antioxidant, antibacterial, and herbicidal agents, showing application potential in food and cosmetics industries, and organic farming agriculture.

SWEET genes encode sugar transporter proteins and often function as susceptibility (S) genes. Consequently, the recessive alleles of these SWEET genes provide resistance. This review summarizes the available literature on the molecular basis of the role of SWEET genes (as S genes) in the host and corresponding transcription activator‐like effectors (TALEs) secreted by the pathogen. The review has four major sections, which follow a brief introduction: The first part gives some details about the occurrence and evolution of SWEET genes in approximately 30 plant species; the second part gives some details about systems where (a) SWEET genes with and without TALEs and (b) TALEs without SWEET genes cause different diseases; the third part summarizes the available information about TALEs along with interfering/truncated TALEs secreted by the pathogens; this section also summarizes the available information on effector‐binding elements (EBEs) available in the promoters of either the SWEET genes or the Executor R genes; the code that is used for binding of TALEs to EBEs is also described in this section; the fourth part gives some details about the available approaches that are being used or can be used in the future for exploiting SWEET genes for developing disease‐resistant cultivars. The review concludes with a section giving conclusions and future possibilities of using SWEET genes for developing disease‐resistant cultivars using different approaches, including conventional breeding and genome editing., SWEET genes function as susceptibility genes. They are activated by binding of TAL effectors (secreted by the pathogen) following a code and can be exploited for developing resistant cultivars.

Phytoalexins play a pivotal role in plant-pathogen interactions. Whereas leaves of rice (Oryza sativa) cultivar Nipponbare predominantly accumulated the phytoalexin sakuranetin after jasmonic acid induction, only very low amounts accumulated in the Kasalath cultivar. Sakuranetin is synthesized from naringenin by naringenin 7-O-methyltransferase (NOMT). Analysis of chromosome segment substitution lines and backcrossed inbred lines suggested that NOMT is the underlying cause of differential phytoalexin accumulation between Nipponbare and Kasalath. Indeed, both NOMT expression and NOMT enzymatic activity are lower in Kasalath than in Nipponbare. We identified a proline to threonine substitution in Kasalath relative to Nipponbare NOMT as the main cause of the lower enzymatic activity. Expanding this analysis to rice cultivars with varying amounts of sakuranetin collected from around the world showed that NOMT induction is correlated with sakuranetin accumulation. In bioassays with Pyricularia oryzae, Gibberella fujikuroi, Bipolaris oryzae, Burkholderia glumae, Xanthomonas oryzae, Erwinia chrysanthemi, Pseudomonas syringae, and Acidovorax avenae, naringenin was more effective against bacterial pathogens and sakuranetin was more effective against fungal pathogens. Therefore, the relative amounts of naringenin and sakuranetin may provide protection against specific pathogen profiles in different rice-growing environments. In a dendrogram of NOMT genes, those from low-sakuranetin-accumulating cultivars formed at least two clusters, only one of which involves the proline to threonine mutation, suggesting that the low sakuranetin chemotype was acquired more than once in cultivated rice. Strains of the wild rice species Oryza rufipogon also exhibited differential sakuranetin accumulation, indicating that this metabolic diversity predates rice domestication.

NLR (Nucleotide-binding [NB] Leucine-rich repeat [LRR] Receptor) proteins are critical for inducing immune responses in response to pathogen proteins, and must be tightly regulated to prevent spurious activation in the absence of a pathogen. The ZAR1 NLR recognizes diverse effector proteins from Pseudomonas syringae, including HopZ1a, and Xanthomonas species. Receptor-like cytoplasmic kinases (RLCKs) such as ZED1, interact with ZAR1 and provide specificity for different effector proteins, such as HopZ1a. We previously developed a transient expression system in Nicotiana benthamiana, that allowed us to demonstrate ZAR1 function is conserved from the Brassicaceae to the Solanaceae. Here, we combined structural modeling of ZAR1, with molecular and functional assays in our transient system, to show that multiple intramolecular and intermolecular interactions regulate ZAR1 activity. We identified new determinants required for the formation of the ZARCC dimer and its activity. Lastly, we characterized new intramolecular interactions between ZAR1 subdomains that participate in keeping ZAR1 immune complexes inactive. This work identifies molecular constraints on immune receptor function and activation.One sentence-summaryStructure-informed analyses reveal multiple finely-tuned intramolecular interactions that regulate the activity of the immune receptor ZAR1.FundingResearch on plant immunity in the Lewis laboratory was supported by the USDA ARS 2030-21000-046-00D and 2030-21000-050-00D (JDL), and the NSF Directorate for Biological Sciences IOS-1557661 (JDL). ECM and AJP acknowledge financial support from UEFISCDI grant PN-III-ID-PCE-2016-0650 and the Romanian Academy programs 1 & 2 of IBAR.

Background Myricetin and sulfonamide derivatives exhibited a wide variety of biological activity. In order to develop highly bioactive molecules, novel myricetin derivatives containing sulfonamide moiety were synthesized and antibacterial activities were investigated. Results The results of bioassays indicated that compound A12, having an EC50 value of 4.7 μg mL-1 , exhibited the best in vitro antibacterial activities against Xanthomonas oryzae pv. oryzae (X. oryzae pv. o.); EC50 values for this compound were even better than those of thiodiazole-copper (TC, 71.4 μg mL-1 ) and bismerthiazol (BT, 54.7 μg mL-1 ). Compound A2, having an EC50 value of 1.1 μg mL-1 , exhibited the best in vitro antibacterial activities against Xanthomonas axonopodis pv. citri (X. axonopodis pv. c); values were notably better than those of TC (60.0 μg mL-1 ) and BT (48.9 μg mL-1 ). Scanning electron microscopy analysis indicated that compounds A2 and A12 caused the cell membranes of X. axonopodis pv. c and X. oryzae pv. o. to break or deform, respectively. When the concentration of compound A12 was 100 μg mL-1 , the effective curative activity against bacterial leaf blight of rice was 44.2% in vivo and the effective protection activity was 58.2% in vivo, results that were both better than values for TC (18.9 and 21.4%, respectively) and BT (12.5 and 12.5%, respectively). Conclusion Novel myricetin derivatives containing a sulfonamide moiety were synthesized and bioassay results showed that compounds A2 and A12 exhibited the best antibacterial activities. © 2019 Society of Chemical Industry.

Summary Transcription Activator‐Like effectors (TALes) represent the largest family of type III effectors among pathogenic bacteria and play a critical role in the process of infection. Strains of Xanthomonas oryzae pv. oryzae (Xoo) and some strains of other Xanthomonas pathogens contain large numbers of TALe genes. Previous techniques to clone individual or a complement of TALe genes through conventional strategies are inefficient and time‐consuming due to multiple genes (up to 29 copies) in a given genome, and technically challenging due to the repetitive sequences (up to 33 nearly identical 102‐nucleotide repeats) of individual TALe genes. Thus, only a limited number of TALe genes have been molecularly cloned and characterized, and the functions of most TALe genes remain unknown. Here, we present an easy and efficient cloning technique to clone TALe genes selectively through in vitro homologous recombination and single‐strand annealing, and demonstrate the feasibility of this approach with four different Xoo strains. Based on the Gibson assembly strategy, two complementary vectors with scaffolds that can preferentially capture all TALe genes from a pool of genomic fragments were designed. Both vector systems enabled cloning of a full complement of TALe genes from each of four Xoo strains and functional analysis of individual TALes in rice in approximately 1 month compared to 3 months by previously used methods. The results demonstrate a robust tool to advance TALe biology and a potential for broad usage of this approach to clone multiple copies of highly competitive DNA elements in any genome of interest.

Summary Poly(A) tail shortening is a critical step in messenger RNA (mRNA) decay and control of gene expression. The carbon catabolite repressor 4 (CCR4)‐associated factor 1 (CAF1) component of the CCR4‐NOT deadenylase complex plays an essential role in mRNA deadenylation in most eukaryotes. However, while CAF1 has been extensively investigated in yeast and animals, its role in plants remains largely unknown. Here, we show that the Citrus sinensis CAF1 (CsCAF1) is a magnesium‐dependent deadenylase implicated in resistance against the citrus canker bacteria Xanthomonas citri. CsCAF1 interacted with proteins of the CCR4‐NOT complex, including CsVIP2, a NOT2 homologue, translin‐associated factor X (CsTRAX) and the poly(A)‐binding proteins CsPABPN and CsPABPC. CsCAF1 also interacted with PthA4, the main X. citri effector required for citrus canker elicitation. We also present evidence suggesting that PthA4 inhibits CsCAF1 deadenylase activity in vitro and stabilizes the mRNA encoded by the citrus canker susceptibility gene CsLOB1, which is transcriptionally activated by PthA4 during canker formation. Moreover, we show that an inhibitor of CsCAF1 deadenylase activity significantly enhanced canker development, despite causing a reduction in PthA4‐dependent CsLOB1 transcription. These results thus link CsCAF1 with canker development and PthA4‐dependent transcription in citrus plants.

Auxin/indole-3-acetic acid proteins (Aux/IAAs) play important roles in auxin signaling pathways, with extensive involvement in plant development and plant response to abiotic and biotic stresses. Manihot esculenta (Cassava) is one of the most important biomass energy crops in tropical regions; however, the information about Aux/IAA proteins remain limited in cassava. In this study, 37 MeAux/IAA gene family members were identified in cassava and a phylogenetic analysis was performed. The transcript levels of MeAux/IAAs were commonly regulated by the pathogen Xanthomonas axonopodis pv manihotis (Xam), and some of them were specifically localized to the nucleus. Moreover, the overexpression of MeAux/IAAs confers an improved disease resistance against Xam in Nicotiana benthamiana, while MeAux/IAAs-silenced plants show disease sensitivity against Xam in cassava, as evidenced by the leaf phenotype and leaf bacterial population. Consistent with the disease resistance, MeAux/IAAs regulated the transcript levels of PATHOGENESIS-RELATED GENES (MePRs), reactive oxygen species accumulation and callose development in the plants' defense response. Taken together, gene profile and functional analysis identified several MeAux/IAAs as novel members in plant disease resistance, providing important information for further utilization of MeAux/IAAs.

Summary The type III secretion system (T3SS) is required for Xanthomonas citri subsp. citri (Xcc) virulence by translocating effectors into host cytoplasm to promote disease development. The T3SS is controlled by the master transcriptional regulators HrpG and HrpX. While the function of HrpG and HrpX are well characterized, their upstream regulation remains elusive. By using transposon mutagenesis, we identified XAC3052, a TetR‐family transcriptional regulator, which regulates T3SS gene expression. Deletion of XAC3052 caused significant reduction in the expression of T3SS and effector genes in vitro and in planta; as well as reduction of virulence in sweet orange (Citrus sinensis). Overexpression of hrpG restored the virulence of ∆XAC3052, suggesting that the loss of virulence is caused by reduction of T3SS gene expression. XAC3052 directly binds to the promoter region and represses the transcription of fadE, mhpC and fadH genes. FadE, MhpC and FadH are not involved in T3SS regulation, but involved in fatty acid catabolism. ∆XAC3052 displays altered fatty acid composition and retarded growth in environments limited in fatty acids. Exogenously supplemented long‐chain fatty acids activate the fadE/mhpC promoter and suppress T3SS promoters in wild‐type Xac but not in ∆XAC3052. Moreover, the binding of XAC3052 to its target promoter was disrupted by long‐chain fatty acids in vitro. Herein, XAC3052 is designated as TfmR (T3SS and Fatty acid Mechanism Regulator). This study identifies a novel regulator of fatty acid metabolism and suggests that fatty acids play an important role in the metabolic control of virulence in Xcc.

Flavin-dependent halogenases selectively introduce halogen substituents into (hetero-)aromatic substrates and require only molecular oxygen and halide salts for this regioselective oxidative CH-functionalization. Genomic analysis of Xanthomonas campestris pv. campestris B100 identified three novel putative members of this enzyme class. They were shown to introduce halogen substituents into, e. g., substituted indoles, while preferring bromide over chloride.

Summary The cucurbit pathogenic bacterium Acidovorax citrulli requires a functional type III secretion system (T3SS) for pathogenicity. In this bacterium, as with Xanthomonas and Ralstonia spp., an AraC‐type transcriptional regulator, HrpX, regulates expression of genes encoding T3SS components and type III‐secreted effectors (T3Es). The annotation of a sequenced A. citrulli strain revealed 11 T3E genes. Assuming that this could be an underestimation, we aimed to uncover the T3E arsenal of the A. citrulli model strain, M6. Thorough sequence analysis revealed 51 M6 genes whose products are similar to known T3Es. Furthermore, we combined machine learning and transcriptomics to identify novel T3Es. The machine‐learning approach ranked all A. citrulli M6 genes according to their propensity to encode T3Es. RNA‐Seq revealed differential gene expression between wild‐type M6 and a mutant defective in HrpX: 159 and 28 genes showed significantly reduced and increased expression in the mutant relative to wild‐type M6, respectively. Data combined from these approaches led to the identification of seven novel T3E candidates that were further validated using a T3SS‐dependent translocation assay. These T3E genes encode hypothetical proteins that seem to be restricted to plant pathogenic Acidovorax species. Transient expression in Nicotiana benthamiana revealed that two of these T3Es localize to the cell nucleus and one interacts with the endoplasmic reticulum. This study places A. citrulli among the ‘richest’ bacterial pathogens in terms of T3E cargo. It also revealed novel T3Es that appear to be involved in the pathoadaptive evolution of plant pathogenic Acidovorax species.

Xanthomonas oryzae pv. oryzae (Xoo) causes a damaging bacterial leaf blight disease in rice. Cold shock proteins (Csps) are highly conserved nucleic acid-binding proteins present in various bacterial genera, but relatively little is known about their functions in Xanthomonas. Herein, we identified four Csps (CspA-CspD) in the Xoo PXO99A strain. Deletion of cspA decreased cold adaptation and a few known pathogenic factors, including bacterial pathogenicity, biofilm formation and polysaccharide production. Furthermore, we performed transcriptomic and chromosome immunoprecipitation (ChIP) experiments to identify direct targets of CspA and to determine its DNA-binding sequence. Integrative data analysis revealed that CspA directly regulates two genes, PXO_RS11830 and PXO_RS01060, by binding to a conserved CCAAT sequence in the promoter region. We generated single-deletion mutants of each gene and the results indicate that both are responsible for Xanthomonas pathogenicity. In addition, quantitative real-time polymerase chain reaction and western blotting showed that CspA suppressed the expression of its direct targets. In summary, our study clarifies the characteristics of Csps in Xanthomonas and greatly advances our understanding of the mechanisms underlying the contribution of CspA to bacterial virulence.

The type III secretion (T3S) system, an essential pathogenicity factor in most Gram-negative plant-pathogenic bacteria, injects bacterial effector proteins directly into the plant cell cytosol. Here, the type III effectors (T3Es) manipulate host cell processes to suppress defence and establish appropriate conditions for bacterial multiplication in the intercellular spaces of the plant tissue. T3E export depends on a secretion signal which is also present in 'non-effectors'. The latter are secreted extracellular components of the T3S apparatus, but are not translocated into the plant cell. How the T3S system discriminates between T3Es and non-effectors is still enigmatic. Previously, we have identified a putative translocation motif (TrM) in several T3Es from Xanthomonas campestris pv. vesicatoria (Xcv). Here, we analysed the TrM of the Xcv effector XopB in detail. Mutation studies showed that the proline/arginine-rich motif is required for efficient type III-dependent secretion and translocation of XopB and determines the dependence of XopB transport on the general T3S chaperone HpaB. Similar results were obtained for other effectors from Xcv. As the arginine residues of the TrM mediate specific binding of XopB to cardiolipin, one of the major lipid components in Xanthomonas membranes, we assume that the association of T3Es to the bacterial membrane prior to secretion supports type III-dependent export.