Identification of Key Transcription Factors Related to Bacterial Spot Resistance in Pepper through Regulatory Network Analyses.

Bacterial spot (BS), caused by Xanthomonas campestris pv. Vesicatoria (Xcv), severely affects the quality and yield of pepper. Thus, breeding new pepper cultivars with enhanced resistance to BS can improve economic benefits for pepper production. Identification of BS resistance genes is an essential step to achieve this goal. However, very few BS resistance genes have been well characterized in pepper so far. In this study, we reanalyzed public multiple time points related to RNA-seq data sets from two pepper cultivars, the Xcv-susceptible cultivar ECW and the Xcv-resistant cultivar VI037601, post Xcv infection. We identified a total of 3568 differentially expressed genes (DEGs) between two cultivars post Xcv infection, which were mainly involved in some biological processes, such as Gene Ontology (GO) terms related to defense response to bacterium, immune system process, and regulation of defense response, etc. Through weighted gene co-expression network analysis (WGCNA), we identified 15 hub (Hub) transcription factor (TF) candidates in response to Xcv infection. We further selected 20 TFs from the gene regulatory network (GRN) potentially involved in Xcv resistance response. Finally, we predicted 4 TFs, C3H (p-coumarate 3-hydroxylase), ERF (ethylene-responsive element binding factor), TALE (three-amino-acid-loop-extension), and HSF (heat shock transcription factor), as key factors responsible for BS disease resistance in pepper. In conclusion, our study provides valuable resources for dissecting the underlying molecular mechanism responsible for Xcv resistance in pepper. Additionally, it also provides valuable references for mining transcriptomic data to identify key candidates for disease resistance in horticulture crops.

Transcriptome analysis reveals defense-related genes and pathways against Xanthomonas campestris pv. vesicatoria in pepper (Capsicum annuum L.).

Bacterial spot (BS), incited by Xanthomonas campestris pv. vesicatoria (Xcv), is one of the most serious diseases of pepper. For a comparative analysis of defense responses to Xcv infection, we performed a transcriptomic analysis of a susceptible cultivar, ECW, and a resistant cultivar, VI037601, using the HiSeqTM 2500 sequencing platform. Approximately 120.23 G clean bases were generated from 18 libraries. From the libraries generated, a total of 38,269 expressed genes containing 11,714 novel genes and 11,232 differentially expressed genes (DEGs) were identified. Functional enrichment analysis revealed that the most noticeable pathways were plant-pathogen interaction, MAPK signaling pathway-plant, plant hormone signal transduction and secondary metabolisms. 1,599 potentially defense-related genes linked to pattern recognition receptors (PRRs), mitogen-activated protein kinase (MAPK), calcium signaling, and transcription factors may regulate pepper resistance to Xcv. Moreover, after Xcv inoculation, 364 DEGs differentially expressed only in VI037601 and 852 genes in both ECW and VI037601. Many of those genes were classified as NBS-LRR genes, oxidoreductase gene, WRKY and NAC transcription factors, and they were mainly involved in metabolic process, response to stimulus and biological regulation pathways. Quantitative RT-PCR of sixteen selected DEGs further validated the RNA-seq differential gene expression analysis. Our results will provide a valuable resource for understanding the molecular mechanisms of pepper resistance to Xcv infection and improving pepper resistance cultivars against Xcv.

Plasma activated water as resistance inducer against bacterial leaf spot of tomato.

Plant bacterial diseases are routinely managed with scheduled treatments based on heavy metal compounds or on antibiotics; to reduce the negative environmental impact due to the use of such chemical compounds, as pollution or selection of antibiotic resistant pathogens, the integrated control management is required. In the frame of a sustainable agriculture the use of bacterial antagonists, biological agents, plant defence response elicitors or resistant host plant genotypes are the most effective approaches. In this work, cold atmospheric pressure plasma (CAP) was applied to sterile distilled water, inducing the production of a hydrogen peroxide, nitrite and nitrate, and a pH reduction. In particular, an atmospheric pressure dielectric barrier discharge (DBD) has been used to produce plasma activated water (PAW), that was firstly assayed in in vitro experiments and then in planta through application at the root apparatus of tomato plants, against Xanthomonas vesicatoria (Xv), the etiological agent of bacterial leaf spot. Moreover, the transcription abundance of five genes related to the plant defense was investigated in response to PAW treatment. PAW did not show direct antimicrobial activity against Xv in in vitro experiments, but it enhanced the tomato plants defenses. It was effective in reducing the disease severity by giving relative protections of ca. 61, 51 and 38% when applied 1 h, 24 h and 6 days before the experimental inoculation, respectively. In addition, the experiments highlighted the pal gene involvement in response to the PAW treatments and against the pathogen; its transcription levels resulted significantly high from 1 to 48 h until their decrease 192 h after PAW application.

Effectiveness of tailocins produced by Pseudomonas fluorescens SF4c in controlling the bacterial-spot disease in tomatoes caused by Xanthomonas vesicatoria.

The development of alternatives for the use of chemical pesticides for plant disease control is the present-day and ongoing challenge for achieving sustainable agriculture. Pseudomonas fluorescens SF4c, native strain from wheat, produces tailocins (phage-tail-like bacteriocins) with antimicrobial activity against several phytopathogenic strains. We thus investigated the efficacy of foliar application of these bacteriocins to control the bacterial-spot disease in tomato caused by Xanthomonas vesicatoria Xcv Bv5-4a. The disease severity and incidence index were reduced by 44 and 36%, respectively; while the number of viable cells of X. vesicatoria Xcv Bv5-4a decreased after bacteriocin treatment. Furthermore, bacteriocin was effective in reducing bacterial-spot-disease symptoms on tomato fruits even when applied 12 h after infection. Tailocin activity was not affected by abiotic influences such as adjuvant, light and temperature and, biotic factors such as apoplastic-fluids. In contrast, no antibacterial activity of these tailocins was observed when the bacteriocin was exposed to extremely dry conditions. Finally, that no cytotoxic effects on mammalian cells were observed with this representative tailocins is highly significant and demonstrates the safety of such compounds in humans. All these findings indicate that the SF4c tailocins represent an attractive alternative to copper-containing bactericides for use in the control of bacterial spot.

Functional Analysis of the Ferric Uptake Regulator Gene fur in Xanthomonas vesicatoria.

Iron is essential for the growth and survival of many organisms. Intracellular iron homeostasis must be maintained for cell survival and protection against iron toxicity. The ferric uptake regulator protein (Fur) regulates the high-affinity ferric uptake system in many bacteria. To investigate the function of the fur gene in Xanthomonas vesicatoria (Xv), we generated a fur mutant strain, fur-m, by site-directed mutagenesis. Whereas siderophore production increased in the Xv fur mutant, extracellular polysaccharide production, biofilm formation, swimming ability and quorum sensing signals were all significantly decreased. The fur mutant also had significantly reduced virulence in tomato leaves. The above-mentioned phenotypes significantly recovered when the Xv fur mutation allele was complemented with a wild-type fur gene. Thus, Fur either negatively or positively regulates multiple important physiological functions in Xv.

Functional and proteomic analyses reveal that wxcB is involved in virulence, motility, detergent tolerance, and biofilm formation in Xanthomonas campestris pv. vesicatoria.

The bacterial envelope possesses diverse functions, including protection against environmental stress and virulence factors for host infection. Here, we report the function of wxcB in Xanthomonas campestris pv. vesicatoria (Xcv), a causal agent of bacterial leaf spot disease in tomato and pepper. To characterize roles of wxcB, we generated a knockout mutant (XcvΔwxcB) and found that the virulence of the mutant was weaker than that of the wild type in tomato plants. To predict the mechanism affected by wxcB, we compared protein expressions between the wild type and the mutant. Expression of 152 proteins showed a greater than 2-fold difference. Proteins involved in motility and cell wall/membrane were the most abundant. Through phenotypic assays, we further demonstrated that the mutant displayed reduced motility and tolerance to treatment, but it showed increased biofilm formation. Interestingly, the LPS profile was unchanged. These results lead to new insights into the functions of wxcB that is associated with cell wall/membrane functions, which contributes to pathogen virulence.

Characterization of HrpB2 from Xanthomonas campestris pv. vesicatoria identifies protein regions that are essential for type III secretion pilus formation.

The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to translocate effector proteins into plant cells. T3S depends on HrpB2, which is essential for assembly of the extracellular T3S pilus and is itself weakly secreted. To characterize the role of HrpB2, we used a transposon mutagenesis approach, which led to the insertion of pentapeptide-encoding sequences into hrpB2. Complementation studies with HrpB2 mutant derivatives revealed that the N-terminal region of HrpB2 tolerates pentapeptide insertions, whereas insertions in the regions spanning amino acids 60-74 and 93-130, respectively, resulted in a loss of bacterial pathogenicity and T3S, including secretion of HrpB2 itself. The C-terminal region (amino acids 93-130) of HrpB2 contains a conserved VxTLxK amino acid motif that is also present in predicted inner rod proteins from animal-pathogenic bacteria and is required for the contribution of HrpB2 to pilus assembly and T3S. Electron microscopy and fractionation studies revealed that HrpB2 is not a component of the extracellular pilus structure but localizes to the bacterial periplasm and the outer membrane. We therefore propose that the essential contribution of HrpB2 to T3S and pilus assembly is linked to its possible function as a periplasmic component of the T3S system at the base of the pilus.

Expression of Xanthomonas campestris pv. vesicatoria type III effectors in yeast affects cell growth and viability.

The gram-negative bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of spot disease in tomato and pepper. X. campestris pv. vesicatoria pathogenicity depends on a type III secretion system delivering effector proteins into the host cells. We hypothesized that some X. campestris pv. vesicatoria effectors target conserved eukaryotic cellular processes and examined phenotypes induced by their expression in yeast. Out of 21 effectors tested, 14 inhibited yeast growth in normal or stress conditions. Viability assay revealed that XopB and XopF2 attenuated cell proliferation, while AvrRxo1, XopX, and XopE1 were cytotoxic. Inspection of morphological features and DNA content of yeast cells indicated that cytotoxicity caused by XopX and AvrRxo1 was associated with cell-cycle arrest at G0/1. Interestingly, XopB, XopE1, XopF2, XopX, and AvrRxo1 that inhibited growth in yeast also caused phenotypes, such as chlorosis and cell death, when expressed in either host or nonhost plants. Finally, the ability of several effectors to cause phenotypes in yeast and plants was dependent on their putative catalytic residues or localization motifs. This study supports the use of yeast as a heterologous system for functional analysis of X. campestris pv. vesicatoria type III effectors, and sets the stage for identification of their eukaryotic molecular targets and modes of action.

CaWRKY2, a chili pepper transcription factor, is rapidly induced by incompatible plant pathogens.

WRKY family proteins are a class of plant-specific transcription factors involved in stress response signaling pathways. In this study a gene encoding a putative WRKY protein was isolated from a pepper EST database (http://genepool.kribb.re.kr). The cDNA, named Capsicum annuum WRKY2 (CaWRKY2), encodes a putative polypeptide of 548 amino acids, containing two WRKY domains with zinc finger motifs and two potential nuclear localization signals. Northern blot analyses showed that CaWRKY2 mRNA was preferentially induced during incompatible interactions of pepper plants with PMMoV, Pseudomonas syringae pv. syringae 61, and Xanthomonas axonopodis pv. vesicatoria race 3. Furthermore, CaWRKY2 transcripts were strongly induced by wounding and ethephon treatment, whereas only moderate expression was detected following treatment with salicylic acid and jasmonic acid. CaWRKY2 was translocated to the nucleus when a CaWRKY2-smGFP fusion construct was expressed in onion epidermal cells. CaWRKY2 also had transcriptional activation activity in yeast. Taken together our data suggest that CaWRKY2 is a pathogen-inducible transcription factor that may have a role in early defense responses to biotic and abiotic stresses.

Physical delimitation of the pepper Bs3 resistance gene specifying recognition of the AvrBs3 protein from Xanthomonas campestris pv. vesicatoria.

The pepper (Capsicum annuum) Bs3 gene confers resistance to avrBs3-expressing strains of the bacterial spot pathogen Xanthomonas campestris pv. vesicatoria. To physically delimit Bs3, a pepper YAC library was screened with two flanking DNA markers that are separated from Bs3 by 1.0 and 1.2 cM, respectively resulting in the identification of three YAC clones. Genetic mapping of the corresponding YACends revealed however, that these YACs do not cover Bs3 and subsequent screens with newly developed YACend markers failed to identify new YAC clones. Marker saturation at the Bs3 locus was carried out by amplified fragment length polymorphism (AFLP). The analysis of 1,024 primer combinations resulted in the identification of 47 new Bs3-linked AFLPs. High-resolution linkage mapping of Bs3 was accomplished by inspecting more than 4,000 F(2) segregants resulting in a genetic resolution of 0.01 cM. Using tightly Bs3-linked YACend- and AFLP-derived markers we established a Bs3-spanning BAC contig and physically delimited the target gene within one BAC clone. The analysis of the Bs3-containing genomic region revealed substantial local variation in the correlation of genetic and physical distances.