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Transcription activator-like effectors (TALEs) are bacterial proteins that are injected into the eukaryotic nucleus to act as transcriptional factors and function as key virulence factors of the phytopathogen Xanthomonas. TALEs are translocated into plant host cells via the type III secretion system and induce the expression of host susceptibility (S) genes to facilitate disease. The unique modular DNA binding domains of TALEs comprise an array of nearly identical direct repeats that enable binding to DNA targets based on the recognition of a single nucleotide target per repeat. The very nature of TALE structure and function permits the proliferation of TALE genes and evolutionary adaptations in the host to counter TALE function, making the TALE−host interaction the most dynamic story in effector biology. The TALE genes appear to be a relatively young effector gene family, with a presence in all virulent members of some species and absent in others. Genome sequencing has revealed many TALE genes throughout the xanthomonads, and relatively few have been associated with a cognate S gene. Several species, including Xanthomonas oryzae pv. oryzae and X. citri pv. citri, have near absolute requirement for TALE gene function, while the genes appear to be just now entering the disease interactions with new fitness contributions to the pathogens of tomato and pepper among others. Deciphering the simple and effective DNA binding mechanism also has led to the development of DNA manipulation tools in fields of gene editing and transgenic research. In the three decades since their discovery, TALE research remains at the forefront of the study of bacterial evolution, plant−pathogen interactions, and synthetic biology. We also discuss critical questions that remain to be addressed regarding TALEs.

Bacterial wilt and canker caused by Clavibacter michiganensis (Cm) inflict considerable damage in tomato-growing regions around the world. Cm has a narrow host range and can cause disease in tomato but not in many eggplant varieties. The pathogenicity of Cm is dependent on secreted serine proteases, encoded by the chp/tomA pathogenicity island (PI), and the pCM2 plasmid. Screening combinations of PI deletion mutants and plasmid-cured strains found that Cm-mediated hypersensitive response (HR) in the Cm-resistant eggplant variety Black Queen is dependent on the chp/tomA PI. Singular reintroduction of PI-encoded serine proteases into Cm

Transcription Activator-Like Effectors are key virulence factors of Xanthomonas. They are secreted into host plant cells and mimic transcription factors inducing the expression of host susceptibility (S) genes. In citrus, CsLOB1 is a direct target of PthA4, the primary effector associated with citrus canker symptoms. CsLOB1 is a transcription factor, and its expression is required for canker symptoms induced by Xanthomonas citri subsp. citri. Several genes are up-regulated by PthA4, however, only CsLOB1 was described as S gene induced by PthA4. Here, we investigated whether other up-regulated genes could be direct targets of PthA4 or CsLOB1. Seven up-regulated genes by PthA4 were investigated, however an expansin-coding gene was more induced than CsLOB1. In Nicotiana benthamiana transient expression experiments, we demonstrate that the expansin-coding gene, referred here to as CsLOB1-INDUCED EXPANSIN 1 (CsLIEXP1), is not a direct target of PthA4, but CsLOB1. Interestingly, CsLIEXP1 was induced by CsLOB1 even without the predicted CsLOB1 binding site, which suggested that CsLOB1 has other unknown binding sites. We also investigated the minimum promoter regulated by CsLOB1, and this region and LOB1 domain were conserved among citrus species and relatives, which suggests that the interaction PthA4-CsLOB1-CsLIEXP1 is conserved in citrus species and relatives. This is the first study that experimentally demonstrated a CsLOB1 downstream target and lay the foundation to identify other new targets. In addition, we demonstrated that the CsLIEXP1 is a putative S gene indirectly induced by PthA4, which may serve as the target for genome editing to generate citrus canker resistant varieties.

Various Gram-negative bacteria use secretion systems to secrete effector proteins that manipulate host biochemical pathways to their benefit. We and others have previously developed machine-learning algorithms to predict novel effectors. Specifically, given a set of known effectors and a set of known non-effectors, the machine-learning algorithm extracts features that distinguish these two protein groups. In the training phase, the machine learning learns how to best combine the features to separate the two groups. The trained machine learning is then applied to open reading frames (ORFs) with unknown functions, resulting in a score for each ORF, which is its likelihood to be an effector. We developed Effectidor, a web server for predicting type III effectors. In this book chapter, we provide a step-by-step introduction to the application of Effectidor, from selecting input data to analyzing the obtained predictions.

Summary The bacterial stringent response is a response to nutrition deprivation and other stress conditions. In Gram‐negative bacteria, this process is mediated by the small signal molecules guanosine pentaphosphate pppGpp and guanosine tetraphosphate ppGpp (collectively referred to as (p)ppGpp), and the RNA polymerase‐binding transcription factor DksA. The (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthase/hydrolase SpoT are responsible for cellular (p)ppGpp levels. Here, we investigated the roles of DksA and (p)ppGpp in the virulence traits of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker. ΔdksA and (p)ppGpp‐deficient ΔspoTΔrelA strains caused reduced virulence and compromised growth in host plants, indicating that DksA and (p)ppGpp are required for full virulence of Xcc. To characterize the effect of stringent response regulators on gene expression, RNA‐seq was conducted using ΔdksA and ΔspoTΔrelA mutant strains grown in hrp‐inducing XVM2 medium. Transcriptome analyses showed that DksA and (p)ppGpp repressed the expression of genes encoding tRNAs, ribosome proteins, iron acquisition and flagellum assembly, and enhanced the expression of genes for histidine metabolism, type 3 secretion system (T3SS), type 2 secretion system (T2SS) and TonB‐dependent transporters. Phenotypically, the ΔdksA and ΔspoTΔrelA strains displayed altered motility, enhanced siderophore production and were unable to cause the hypersensitive response on non‐host plants. In conclusion, stringent response regulators DksA and (p)ppGpp play an important role in virulence, nutrition uptake and host adaptation of Xcc.

The temporal expression profiles of citrus leaves explain the sink–source transition of immature leaves to mature leaves and provide knowledge regarding the differential responses of mature and immature leaves to biotic stress such as citrus canker and Asian citrus psyllid (Diaphorina citri). Citrus is an important fruit crop worldwide. Different developmental stages of citrus leaves are associated with distinct features, such as differences in susceptibilities to pathogens and insects, as well as photosynthetic capacity. Here, we investigated the mechanisms underlying these distinctions by comparing the gene expression profiles of mature and immature citrus leaves. Immature (stages V3 and V4), transition (stage V5), and mature (stage V6) Citrus sinensis leaves were chosen for RNA-seq analyses. Carbohydrate biosynthesis, photosynthesis, starch biosynthesis, and disaccharide metabolic processes were enriched among the upregulated differentially expressed genes (DEGs) in the V5 and V6 stages compared with that in the V3 and V4 stages. Glucose level was found to be higher in V5 and V6 than in V3 and V4. Among the four stages, the largest number of DEGs between contiguous stages were identified between V5 and V4, consistent with a change from sink to source, as well as with the sucrose and starch quantification data. The differential expression profiles related to cell wall synthesis, secondary metabolites such as flavonoids and terpenoids, amino acid biosynthesis, and immunity between immature and mature leaves may contribute to their different responses to Asian citrus psyllid infestation. The expression data suggested that both the constitutive and induced gene expression of immunity-related genes plays important roles in the greater resistance of mature leaves against Xanthomonas citri compared with immature leaves. The gene expression profiles in the different stages can help identify stage-specific promoters for the manipulation of the expression of citrus traits according to the stage. The temporal expression profiles explain the sink–source transition of immature leaves to mature leaves and provide knowledge regarding the differential responses to biotic stress.

Transcription activator-like effectors (TALEs) are virulence factors of Xanthomonas that induce the expression of host susceptibility (S) genes by specifically binding to effector binding elements (EBEs) in their promoter regions. The DNA binding specificity of TALEs is dictated by their tandem repeat regions, which are highly variable between different TALEs. Mutation of the EBEs of S genes is being utilized as a key strategy to generate resistant crops against TALE-dependent pathogens. However, TALE adaptations through rearrangement of their repeat regions is a potential obstacle for successful implementation of this strategy. We investigated the consequences of TALE adaptations in the citrus pathogen Xanthomonas citri subsp. citri (Xcc), in which PthA4 is the TALE required for pathogenicity, whereas CsLOB1 is the corresponding susceptibility gene, on host resistance. Seven TALEs, containing two-to-nine mismatching-repeats to the EBEPthA4 that were unable to induce CsLOB1 expression, were introduced into Xcc pthA4:Tn5 and adaptation was simulated by repeated inoculations into and isolations from sweet orange for a duration of 30 cycles. While initially all strains failed to promote disease, symptoms started to appear between 9–28 passages in four TALEs, which originally harbored two-to-five mismatches. Sequence analysis of adapted TALEs identified deletions and mutations within the TALE repeat regions which enhanced putative affinity to the CsLOB1 promoter. Sequence analyses suggest that TALEs adaptations result from recombinations between repeats of the TALEs. Reintroduction of these adapted TALEs into Xcc pthA4:Tn5 restored the ability to induce the expression of CsLOB1, promote disease symptoms and colonize host plants. TALEs harboring seven-to-nine mismatches were unable to adapt to overcome the incompatible interaction. Our study experimentally documented TALE adaptations to incompatible EBE and provided strategic guidance for generation of disease resistant crops against TALE-dependent pathogens., Author summary Mutation of the EBEs of susceptibility (S) genes via genome editing and utilization of naturally occurring EBE variants have been used to generate disease resistant plants. However, TALE adaptations may lead to resistance loss, limiting the long-term efficacy of the strategy. We utilized an experimental evolution approach to test TALEs adaptations in the Xanthomonas citri-citrus pathosystem using designer TALEs that cannot recognize the EBE of host targets. We identified adaptive TALE mutations and deletions that occurred during less than 30 cycles of repeated infections, which reconstituted the virulence on the host. Adaptive variants originated from TALEs that harbored a small number of mismatches (≤5) to the EBE, whereas designer TALEs that harbored larger number of mismatches (≥7) to the EBE failed to adapt in the duration of this study. Our study experimentally demonstrates adaptive rearrangements of TALEs during host adaptation and suggests that the potential durability in the resistance of modified crops should be a significant factor to be considered prior to their introduction into the field.

Bacteria of the genus Xanthomonas cause a wide variety of economically important diseases in most crops. The virulence of the majority of Xanthomonas spp. is dependent on secretion and translocation of effectors by the type 3 secretion system (T3SS) that is controlled by two master transcriptional regulators HrpG and HrpX. Since their discovery in the 1990s, the two regulators were the focal point of many studies aiming to decipher the regulatory network that controls pathogenicity in Xanthomonas bacteria. HrpG controls the expression of HrpX, which subsequently controls the expression of T3SS apparatus genes and effectors. The HrpG/HrpX regulon is activated in planta and subjected to tight metabolic and genetic regulation. In this review, we cover the advances made in understanding the regulatory networks that control and are controlled by the HrpG/HrpX regulon and their conservation between different Xanthomonas spp.

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating diseases in citrus. Meiwa kumquat (Fortunella crassifolia) has shown a durable resistance against Xcc. Here, we aimed to characterize the mechanisms responsible for such a durable resistance by characterizing the transcriptional and physiological responses of Meiwa kumquat to Xcc. Inoculation of Meiwa kumquat with Xcc promoted immune responses such as upregulation of PR genes, accumulation of salicylic acid, hypersensitive response (HR)-like cell death and early leaf abscission. Hypertrophy and hyperplasia symptoms, which are known to be caused by Xcc-induction of the canker susceptibility gene LOB1 through the transcription activator-like effector (TALE) PthA4, always appear prior to the development of cell death. Mutation of pthA4 in Xcc abolished the induction of LOB1, canker symptoms, cell death, and leaf abscission and reduced the expression of PR genes in inoculated kumquat leaves without reducing Xcc titers in planta. Transcriptome analysis demonstrated that PthA4 promotes plant biotic and abiotic stress responses and the biosynthesis of abscisic acid. Transcriptional induction of LOB1 homologs in Meiwa kumquat by Xcc pthA4 mutant strains carrying a repertoire of designer TALEs promoted the elicitation of HR-like phenotype and leaf abscission, suggesting that kumquat response to Xcc is associated with upregulation of LOB1. Our study suggests a novel mechanism of plant resistance to Xanthomonas via elicitation of immune responses by upregulation of a host susceptibility gene., Author summary Kumquats, members of the Citrus genus, are known for their durable resistance against citrus canker disease caused by the plant pathogenic bacterium Xanthomonas citri subsp. citri (Xcc). Here, we characterized the resistance mechanisms of Meiwa kumquat to citrus canker. We demonstrate that Xcc infection of Meiwa kumquat causes both immune responses and early leaf abscission, which together contribute to the resistance of kumquat to the pathogen. In addition, we showed that kumquat immune responses and early leaf abscission in the presence of Xcc are elicited by the transcription activator-like effector PthA4, one of the predominant pathogenicity determinants of Xcc in susceptible citrus varieties. PthA4-mediated elicitation of immune responses and early leaf abscission in kumquat occurs via transcriptional induction of the LOB1 gene, which is associated with disease development in susceptible citrus varieties by promoting hypertrophy and hyperplasia symptoms. LOB1 seems to play a dual role in the Xcc-kumquat interaction by acting as a susceptibility gene by promoting hypertrophy and hyperplasia symptoms in the early stages and as a potential activator of immune responses in the late stages; this poses an insurmountable challenge for the pathogen to overcome, contributing to the durable resistance of kumquat to Xcc.

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.

Xanthomonas citri ssp. citri (Xcc) is an important plant-pathogenic bacterium that causes citrus canker disease worldwide. PthA, a transcriptional activator-like (TAL) effector, directs the expression of the canker susceptibility gene CsLOB1. Here, we report our recent progress in the functional characterization of CsLOB1. Subcellular localization analysis of CsLOB1 protein in citrus protoplast revealed that CsLOB1 is primarily localized in the nucleus. We showed that CsLOB1 expression driven by dexamethasone (DEX) in CsLOB1-GR transgenic plants is associated with pustule formation following treatment with DEX. Pustule formation was not observed in DEX-treated wild-type plants and in non-treated CsLOB1-GR transgenic plants. Water soaking is typically associated with symptoms of citrus canker. Weaker water soaking was observed with pustule formation in CsLOB1-GR transgenic plants following DEX treatment. When CsLOB1-GR-transgenic Duncan grapefruit leaves were inoculated with Xcc306ΔpthA4 and treated with DEX, typical canker symptoms, including hypertrophy, hyperplasia and water soaking symptoms, were observed on DEX-treated transgenic plant leaves, but not on mock-treated plants. Twelve citrus genes that are induced by PthA4 are also stimulated by the DEX-induced expression of CsLOB1. As CsLOB1 acts as a transcriptional factor, we identified putative targets of CsLOB1 via bioinformatic and electrophoretic mobility shift assays. Cs2g20600, which encodes a zinc finger C3HC4-type RING finger protein, has been identified to be a direct target of CsLOB1. This study advances our understanding of the function of CsLOB1 and the molecular mechanism of how Xcc causes canker symptoms via CsLOB1.

Xanthomonas is a well-studied genus of bacterial plant pathogens whose members cause a variety of diseases in economically important crops worldwide. Genomic and functional studies of these phytopathogens have provided significant understanding of microbial-host interactions, bacterial virulence and host adaptation mechanisms including microbial ecology and epidemiology. In addition, several strains of Xanthomonas are important as producers of the extracellular polysaccharide, xanthan, used in the food and pharmaceutical industries. This polymer has also been implicated in several phases of the bacterial disease cycle. In this review, we summarise the current knowledge on the infection strategies and regulatory networks controlling virulence and adaptation mechanisms from Xanthomonas species and discuss the novel opportunities that this body of work has provided for disease control and plant health., Here, we discuss the current knowledge surrounding regulatory networks and systems that control virulence and adaption mechanisms in Xanthomonas species. Additionally, we detail how study of these pathogens has provided novel opportunities for disease control and plant health.

The 14-3-3 phospho-binding proteins with scaffolding activity play central roles in the regulation of enzymes and signaling complexes in eukaryotes. In plants, 14-3-3 isoforms are required for disease resistance and key targets of pathogen effectors. Here, we examined the requirement of the tomato (Solanum lycopersicum) 14-3-3 isoform (TFT) protein family for Xv3 disease resistance in response to the bacterial pathogen Xanthomonas euvesicatoria. In addition, we determined whether TFT proteins interact with the repertoire of X. euvesicatoria type III secretion effector proteins, including AvrXv3, the elicitor of Xv3 resistance. We show that multiple TFT contribute to Xv3 resistance. We also show that one or more TFT proteins physically interact with multiple effectors (AvrXv3, XopE1, XopE2, XopN, XopO, XopQ, and XopAU). Genetic analyses indicate that none of the identified effectors interfere with AvrXv3-elicited resistance into Xv3 tomato leaves; however, XopE1, XopE2, and XopO are required to suppress symptom development in susceptible tomato leaves. Phospho-peptide mapping revealed that XopE2 is phosphorylated at multiple residues in planta and residues T66, T131, and S334 are required for maximal binding to TFT10. Together, our data support the hypothesis that multiple TFT proteins are involved in immune signaling during X. euvesicatoria infection.

Summary The Gram-negative bacterium Xanthomonas euvesicatoria (Xcv) is the causal agent of bacterial spot disease in pepper and tomato. Xcv pathogenicity depends on a type III secretion (T3S) system that delivers effector proteins into host cells to suppress plant immunity and promote disease. The pool of known Xcv effectors includes approximately 30 proteins, most identified in the 85-10 strain by various experimental and computational techniques. To identify additional Xcv 85-10 effectors, we applied a genome-wide machine-learning approach, in which all open reading frames (ORFs) were scored according to their propensity to encode effectors. Scoring was based on a large set of features, including genomic organization, taxonomic dispersion, hypersensitive response and pathogenicity (hrp)-dependent expression, 5′ regulatory sequences, amino acid composition bias and GC content. Thirty-six predicted effectors were tested for translocation into plant cells using the hypersensitive response (HR)-inducing domain of AvrBs2 as a reporter. Seven proteins (XopAU, XopAV, XopAW, XopAP, XopAX, XopAK and XopAD) harboured a functional translocation signal and their translocation relied on the HrpF translocon, indicating that they are bona fide T3S effectors. Remarkably, four belong to novel effector families. Inactivation of the xopAP gene reduced the severity of disease symptoms in infected plants. A decrease in cell death and chlorophyll content was observed in pepper leaves inoculated with the xopAP mutant when compared with the wild-type strain. However, populations of the xopAP mutant in infected leaves were similar in size to those of wild-type bacteria, suggesting that the reduction in virulence was not caused by impaired bacterial growth.

Xanthomonas citri ssp. citri (Xcc) is an important plant‐pathogenic bacterium that causes citrus canker disease worldwide. PthA, a transcriptional activator‐like (TAL) effector, directs the expression of the canker susceptibility gene CsLOB1. Here, we report our recent progress in the functional characterization of CsLOB1. Subcellular localization analysis of CsLOB1 protein in citrus protoplast revealed that CsLOB1 is primarily localized in the nucleus. We showed that CsLOB1 expression driven by dexamethasone (DEX) in CsLOB1‐GR transgenic plants is associated with pustule formation following treatment with DEX. Pustule formation was not observed in DEX‐treated wild‐type plants and in non‐treated CsLOB1‐GR transgenic plants. Water soaking is typically associated with symptoms of citrus canker. Weaker water soaking was observed with pustule formation in CsLOB1‐GR transgenic plants following DEX treatment. When CsLOB1‐GR‐transgenic Duncan grapefruit leaves were inoculated with Xcc306ΔpthA4 and treated with DEX, typical canker symptoms, including hypertrophy, hyperplasia and water soaking symptoms, were observed on DEX‐treated transgenic plant leaves, but not on mock‐treated plants. Twelve citrus genes that are induced by PthA4 are also stimulated by the DEX‐induced expression of CsLOB1. As CsLOB1 acts as a transcriptional factor, we identified putative targets of CsLOB1 via bioinformatic and electrophoretic mobility shift assays. Cs2g20600, which encodes a zinc finger C3HC4‐type RING finger protein, has been identified to be a direct target of CsLOB1. This study advances our understanding of the function of CsLOB1 and the molecular mechanism of how Xcc causes canker symptoms via CsLOB1.

Summary Effector-triggered immunity (ETI) to host-adapted pathogens is associated with rapid cell death at the infection site. The plant-pathogenic bacterium Xanthomonas euvesicatoria (Xcv) interferes with plant cellular processes by injecting effector proteins into host cells through the type III secretion system. Here, we show that the Xcv effector XopQ suppresses cell death induced by components of the ETI-associated MAP kinase cascade MAPKKKα MEK2/SIPK and by several R/avr gene pairs. Inactivation of xopQ by insertional mutagenesis revealed that this effector inhibits ETI-associated cell death induced by avirulent Xcv in resistant pepper (Capsicum annuum), and enhances bacterial growth in resistant pepper and tomato (Solanum lycopersicum). Using protein–protein interaction studies in yeast (Saccharomyces cerevisiae) and in planta, we identified the tomato 14–3–3 isoform SlTFT4 and homologs from other plant species as XopQ interactors. A mutation in the putative 14–3–3 binding site of XopQ impaired interaction of the effector with CaTFT4 in yeast and its virulence function in planta. Consistent with a role in ETI, TFT4 mRNA abundance increased during the incompatible interaction of tomato and pepper with Xcv. Silencing of NbTFT4 in Nicotiana benthamiana significantly reduced cell death induced by MAPKKKα. In addition, silencing of CaTFT4 in pepper delayed the appearance of ETI-associated cell death and enhanced growth of virulent and avirulent Xcv, demonstrating the requirement of TFT4 for plant immunity to Xcv. Our results suggest that the XopQ virulence function is to suppress ETI and immunity-associated cell death by interacting with TFT4, which is an important component of ETI and a bona fide target of XopQ.

Mitogen-activated protein kinase (MAPK) cascades play a fundamental role in signaling of plant immunity and mediate elicitation of cell death. Xanthomonas spp. manipulate plant signaling by using a type III secretion system to deliver effector proteins into host cells. We examined the ability of 33 Xanthomonas effectors to inhibit cell death induced by overexpression of components of MAPK cascades in Nicotiana benthamiana plants. Five effectors inhibited cell death induced by overexpression of MAPKKKα and MEK2, but not of MAP3Ke. In addition, expression of AvrBs1 in yeast suppressed activation of the high osmolarity glycerol MAPK pathway, suggesting that the target of this effector is conserved in eukaryotic organisms. These results indicate that Xanthomonas employs several type III effectors to suppress immunity-associated cell death mediated by MAPK cascades.

The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) causes wilt and canker disease of tomato (Solanum lycopersicum). Mechanisms of Cmm pathogenicity and tomato response to Cmm infection are not well understood. To explore the interaction between Cmm and tomato, multidimensional protein identification technology (MudPIT) and tandem mass spectrometry were used to analyze in vitro and in planta generated samples. The results show that during infection Cmm senses the plant environment, transmits signals, induces, and then secretes multiple hydrolytic enzymes, including serine proteases of the Pat-1, Ppa, and Sbt familes, the CelA, XysA, and NagA glycosyl hydrolases, and other cell wall-degrading enzymes. Tomato induction of pathogenesis-related (PR) proteins, LOX1, and other defense-related proteins during infection indicates that the plant senses the invading bacterium and mounts a basal defense response, although partial with some suppressed components including class III peroxidases and a secreted serine peptidase. The tomato ethylene-synthesizing enzyme ACC-oxidase was induced during infection with the wild-type Cmm but not during infection with an endophytic Cmm strain, identifying Cmm-triggered host synthesis of ethylene as an important factor in disease symptom development. The proteomic data were also used to improve Cmm genome annotation, and thousands of Cmm gene models were confirmed or expanded.

The type III effector HsvG of the gall-forming Pantoea agglomerans pv. gypsophilae is a DNA-binding protein that is imported to the host nucleus and involved in host specificity. The DNA-binding region of HsvG was delineated to 266 amino acids located within a secondary structure region near the N-terminus of the protein but did not display any homology to canonical DNA-binding motifs. A binding site selection procedure was used to isolate a target gene of HsvG, named HSVGT, in Gypsophila paniculata. HSVGT is a predicted acidic protein of the DnaJ family with 244 amino acids. It harbors characteristic conserved motifs of a eukaryotic transcription factor, including a bipartite nuclear localization signal, zinc finger, and leucine zipper DNA-binding motifs. Quantitative real-time polymerase chain reaction analysis demonstrated that HSVGT transcription is specifically induced in planta within 2 h after inoculation with the wild-type P. agglomerans pv. gypsophilae compared with the hsvG mutant. Induction of HSVGT reached a peak of sixfold at 4 h after inoculation and progressively declined thereafter. Gel-shift assay demonstrated that HsvG binds to the HSVGT promoter, indicating that HSVGT is a direct target of HsvG. Our results support the hypothesis that HsvG functions as a transcription factor in gypsophila.

HsvG and HsvB, two paralogous type III effectors of the gall-forming bacteria Pantoea agglomerans pv. gypsophilae and P. agglomerans pv. betae, determine host specificity on gypsophila and beet, respectively. They were previously shown to be DNA-binding proteins imported into host and non-host nuclei and might act as transcriptional activators. Sequence analysis of these effectors did not detect canonical nuclear localization signals (NLSs), but two basic amino acid clusters designated putative NLS1 and NLS2 were detected in their N-terminal and C-terminal regions, respectively. pNIA assay for nuclear import in yeast and bombardment of melon leaves with each of the NLSs fused to a 2xYFP reporter indicated that putative NLS1 and NLS2 were functional in transport of HsvG into the nucleus. A yeast two-hybrid assay showed that HsvB, HsvG, putative NLS1, putative NLS2, HsvG converted into HsvB, or HsvB converted into HsvG by exchanging the repeat domain, all interacted with AtKAP-α and importin-α3 of Arabidopsis thaliana. Deletion analysis of the NLS domains in HsvG suggested that putative NLS1 or NLS2 were required for pathogenicity on gypsophila cuttings and presumably for import of HsvG into the nucleus. This study demonstrates the presence of two functional NLSs in the type III effectors HsvG and HsvB.