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Clavibacter bacteria use secreted apoplastic effectors, such as putative serine proteases, for virulence in host plants and for hypersensitive response (HR) induction in nonhost plants. Previously, we have shown that Clavibacter capsici ChpGCc is important for the necrosis development in pepper (Capsicum annuum) leaves. Here, we determine the function of ChpGCc, along with three paralogous proteins, for HR induction in the apoplastic space of a nonhost plant, Nicotiana tabacum. The full-length and signal peptide–deleted (ΔSP) mature forms of all proteins fused with the tobacco PR1b signal sequence were generated. The full-length and ΔSP forms of ChpGCc and only the ΔSP forms of ChpECc and Pat-1Cc, but none of the ChpCCc, triggered HR. Based on the predicted protein structures, ChpGCc carries amino acids for a catalytic triad and a disulfide bridge in positions like Pat-1Cm. Substituting these amino acids of ChpGCc with alanine abolished or reduced HR-inducing activity. To determine whether these residues are important for necrosis development in pepper, alanine-substituted chpGCc genes were transformed into the C. capsici PF008ΔpCM1 strain, which lacks the intact chpGCc gene. The strain with any variants failed to restore the necrosis-causing ability. These results suggest that ChpGCc has a dual function as a virulence factor in host plants and an HR elicitor in nonhost plants. Based on our findings and previous results, we propose Clavibacter apoplastic effectors, such as ChpGCc, Pat-1Cm, Chp-7Cs, and ChpGCm, as hypersensitive response and virulence (Hrv) proteins that display phenotypic similarities to the hypersensitive response and pathogenicity (Hrp) proteins found in gram-negative bacteria. [Graphic: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Nucleotide-binding leucine-rich repeat receptors (NLRs) are the most abundant type of immune receptors in plants and can trigger a rapid cell-death (hypersensitive) response upon sensing pathogens. We previously cloned the wheat NLR Sr35, which encodes a coiled-coil (CC) NLR that confers resistance to the virulent wheat stem rust race Ug99. Here, we investigated Sr35 signaling after Agrobacterium-mediated transient expression in Nicotiana benthamiana. Expression of Sr35 in N. benthamiana leaves triggered a mild cell-death response, which is enhanced in the autoactive mutant Sr35 D503V. The N-terminal tagging of Sr35 with green fluorescent protein (GFP) blocked the induction of cell death, whereas a C-terminal GFP tag did not. No domain truncations of Sr35 generated cell-death responses as strong as the wild type, but a truncation including the NB-ARC (nucleotide binding adaptor) shared by APAF-1, R proteins, and CED-4 domains in combination with the D503V autoactive mutation triggered cell death. In addition, coexpression of Sr35 with the matching pathogen effector protein AvrSr35 resulted in robust cell death and electrolyte leakage levels that were similar to autoactive Sr35 and significantly higher than Sr35 alone. Coexpression of Sr35-CC-NB-ARC and AvrSr35 did not induce cell death, confirming the importance of the leucine-rich repeat (LRR) domain for AvrSr35 recognition. These findings were confirmed through Agrobacterium-mediated transient expression in barley. Taken together, these results implicate the CC-NB-ARC domains of Sr35 in inducing cell death and the LRR domain in AvrSr35 recognition.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

IntroductionThe type III effector RipAA of Ralstonia solanacearum GMI1000 plays a critical role in the incompatible interaction on Nicotiana benthamiana.MethodsThe RipAA was transiently expressed in N. benthamiana by Agrobacterium-mediated transformation. Chemical staining with trypan blue and DAB were conducted to examine the cell death and the accumulation of hydrogen peroxide (H2O2), respectively. The expression of the marker genes for salicylic acid (SA) and jasmonic acid (JA) signaling was evaluated by quantitative reverse transcription PCR (qRT-PCR). The proteins interacted with RipAA was identified from N. benthamiana by yeast two-hybrid and pull-down assays. A TRV-mediated gene silencing was used to assess the role of host gene in response to RipAA expression and R. solanacearum infection.Results and discussionRipAA induced the accumulation of hydrogen peroxide (H2O2) and genome DNA degradation in N. benthamiana, which were accompanied by a hypersensitive reaction. Simultaneously, the marker genes for salicylic acid (SA) signaling were induced and those for jasmonic acid (JA) signaling were reduced. N. benthamiana chloroplastic AtpB, the ATPase β subunit, was identified as an interactor with RipAA. The silencing of atpB in N. benthamiana resulted in the inability of RipAA to induce a hypersensitive response, a compatible interaction with GMI1000, and an enhanced sensitivity to bacterial wilt. Our data support the concept that RipAA determines host-range specificity by targeting the host chloroplastic AtpB.

Citrus canker disease caused by Xanthomonas citri subsp. citri is one of the most destructive diseases in citrus. X. citri subsp. citri pathotypes display different host ranges. X. citri subsp. citri strain A (XccA) causes canker disease in most commercial citrus varieties, whereas strain AW (XccAW), which is genetically similar to XccA, infects only lime and alemow. Understanding the mechanism that determines the host range of pathogens is critical to investigating and utilizing host resistance. We hypothesized that XccAW would undergo mutations in genes that restrict its host range when artificially inoculated into incompatible citrus varieties. To test this hypothesis, we used an experimental evolution approach to identify phenotypic traits and genetic loci associated with the adaptation of XccAW to incompatible sweet orange. Repeated inoculation and reisolation cycles improved the ability of three independent XccAW strains to colonize sweet orange. Adapted XccAW strains displayed increased expression of type III secretion system and effector genes. Genome sequencing analysis indicated that two of the adapted strains harbored mutations in pthAW1, a transcription activator-like effector (TALE) gene, that corresponded to the removal of one or two repeats from the central DNA-binding repeat region. Introduction of the original but not the adapted pthAW1 variants into XccA abolished its ability to cause canker symptoms in sweet orange, Meyer lemon, and clementine but not in other XccAW-resistant citrus varieties. The original pthAW1, when expressed in XccA, induced ion leakage and the expression of pathogenesis-related genes but had no effect on CsLOB1 expression in sweet orange. Our study has identified a novel host-specific avirulence TALE and demonstrated active adaptive rearrangements of the TALE repeat array during host adaptation.[Graphic: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Grapevine downy mildew is the most serious disease of grapevine cultivars that affects the rate of resistance/susceptibility to Plasmopara viticola. In this study, we used the susceptible cultivar “Zitian Seedless” and the resistant cultivar “Kober 5BB” as materials to determine the transcriptome differences and phenotypes of the leaves after inoculation with downy mildew. The differences in microstructures and molecular levels were compared and analyzed. Fluorescence staining and microscopic observations confirmed that hypersensitive cell death occurred around the stomata in “Kober 5BB” infected by downy mildew zoospores. Meanwhile, transcriptomic profiling indicated that there were 11,713 and 6,997 gene expression differences between the resistant and susceptible cultivars at 72 h after inoculation when compared to control (0 h), respectively. The differentially expressed genes of the two cultivars are significantly enriched in different pathways, including response to plant-pathogen interaction, mitogen-activated protein kinase (MAPK) signaling pathway, plant hormone signal transduction, phenylpropanoid, and flavonoid biosynthesis. Furthermore, the results of functional enrichment analysis showed that H2O2 metabolism, cell death, reactive oxygen response, and carbohydrate metabolism are also involved in the defense response of “Kober 5BB,” wherein a total of 322 key genes have been identified. The protein interaction network showed that metacaspases (MCAs), vacuolar processing enzymes (VPEs), and Papain-like cysteine proteases (PLCPs) play an important role in the execution of hypersensitive responses (HR). In conclusion, we demonstrated that HR cell death is the key strategy in the process of grape defense against downy mildew, which may be mediated or activated by Caspase-like proteases.

CfAvr4, a chitin-binding effector protein produced by the Dothideomycete tomato pathogen Cladosporium fulvum, protects the cell wall of this fungus against hydrolysis by secreted host chitinases during infection. However, in the presence of the Cf-4 immune receptor of tomato, CfAvr4 triggers a hypersensitive response (HR), which renders the pathogen avirulent. Recently, several orthologues of CfAvr4 have been identified from phylogenetically closely related species of Dothideomycete fungi. Of these, DsAvr4 from Dothistroma septosporum also triggers a Cf-4-dependent HR, but CaAvr4 and CbAvr4 from Cercospora apii and Cercospora beticola, respectively, do not. All, however, bind chitin. To identify the region(s) and specific amino acid residue(s) of CfAvr4 and DsAvr4 required to trigger a Cf-4-dependent HR, chimeric and mutant proteins, in which specific protein regions or single amino acid residues, respectively, were exchanged between CfAvr4 and CaAvr4 or DsAvr4 and CbAvr4, were tested for their ability to trigger an HR in Nicotiana benthamiana plants transgenic for the Cf-4 immune receptor gene. Based on this approach, a single region common to CfAvr4 and DsAvr4 was determined to carry a conserved proline residue necessary for the elicitation of this HR. In support of this result, a Cf-4-dependent HR was triggered by mutant CaAvr4 and CbAvr4 proteins carrying an arginine-to-proline substitution at this position. This study provides the first step in deciphering how Avr4 orthologues from different Dothideomycete fungi trigger a Cf-4-dependent HR.

Abstract Background Heat resistance is a common characteristic of harpins, a class of proteins found in Gram-negative bacteria, which may be related to the stability of coiled-coil (CC) structure. The CC structure is a ubiquitous protein folding and assembly motif made of α-helices wrapping around each other forming a supercoil. Specifically, whether the stability of the CC structure near to N-terminus of four selected harpin proteins from Xanthomonas (hereafter referred to as Hpa1) would influence their characteristics of heat resistance was investigated. We used bioinformatics approach to predict the structure of Hpa1, used the performance of hypersensitive response (HR)-induction activity of Hpa1 and circular dichroism (CD) spectral analyses to detect the relationship between the stability of the CC structure of Hpa1 and heat resistance. Results Each of four-selected Hpa1 has two α-helical regions with one in their N-terminus that could form CC structure, and the other in their C-terminus that could not. And the important amino acid residues involved in the CC motifs are located on helices present on the surface of these proteins, indicating they may engage in the formation of oligo mericaggregates, which may be responsible for HR elicitation by harpins and their high thermal stability. Increased or decreased the probability of forming a CC could either induce a stronger HR response or eliminate the ability to induce HR in tobacco after high temperature treatment. In addition, although the four Hpa1 mutants had little effect on the induction of HR by Hpa1, its thermal stability was significantly decreased. The α-helical content increased with increasing temperature, and the secondary structures of Hpa1 became almost entirely α-helices when the temperature reached 200 °C. Moreover, the stability of the CC structure near to N-terminus was found to be positively correlated with the heat resistance of Hpa1. Conclusions The stability of the CC structure might sever as an inner drive for mediating the heat resistance of harpin proteins. Our results offer a new insight into the interpretation of the mechanism involved in the heat resistance of harpin protein and provide a theoretical basis for further harpin function investigations and structure modifications.

Nucleotide-binding leucine-rich repeat receptors (NLRs) are the most abundant type of immune receptors in plants and can trigger a rapid cell-death (hypersensitive) response upon sensing pathogens. We previously cloned the wheat NLR Sr35, which encodes a coiled-coil (CC) NLR that confers resistance to the virulent wheat stem rust race Ug99. Here, we investigated Sr35 signaling after Agrobacterium-mediated transient expression in Nicotiana benthamiana. Expression of Sr35 in N. benthamiana leaves triggered a mild cell-death response, which is enhanced in the autoactive mutant Sr35 D503V. The N-terminal tagging of Sr35 with green fluorescent protein (GFP) blocked the induction of cell death, whereas a C-terminal GFP tag did not. No domain truncations of Sr35 generated cell-death responses as strong as the wild type, but a truncation including the NB-ARC (nucleotide binding adaptor) shared by APAF-1, R proteins, and CED-4 domains in combination with the D503V autoactive mutation triggered cell death. In addition, coexpression of Sr35 with the matching pathogen effector protein AvrSr35 resulted in robust cell death and electrolyte leakage levels that were similar to autoactive Sr35 and significantly higher than Sr35 alone. Coexpression of Sr35-CC-NB-ARC and AvrSr35 did not induce cell death, confirming the importance of the leucine-rich repeat (LRR) domain for AvrSr35 recognition. These findings were confirmed through Agrobacterium-mediated transient expression in barley. Taken together, these results implicate the CC-NB-ARC domains of Sr35 in inducing cell death and the LRR domain in AvrSr35 recognition.[Graphic: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Abstract Background Harpins are proteins secreted by the type III secretion system of Gram-negative bacteria during pathogen–plant interactions that can act as elicitors, stimulating defense and plant growth in many types of non-host plants. Harpin-treated plants have higher resistance, quality and yields and, therefore, harpin proteins may potentially have many valuable agricultural applications. Harpins are characterized by high thermal stability at 100 °C. However, it is unknown whether harpins are still active at temperatures above 100 °C or whether different temperatures affect the activity of the harpin protein in different ways. The mechanism responsible for the heat stability of harpins is also unknown. Results We identified a novel harpin, HpaXpm, from the cassava blight bacteria Xanthomonas phaseoli pv. manihotis HNHK. The predicted secondary structure and 3-D structure indicated that the HpaXpm protein has two β-strand domains and two major α-helical domains located at the N- and C-terminal regions, respectively. A phylogenetic tree generated using the maximum likelihood method grouped HpaXpm in clade I of the Hpa1 group along with harpins produced by other Xanthomonas spp. (i.e., HpaG-Xag, HpaG-Xcm, Hpa1-Xac, and Hpa1Xm). Phenotypic assays showed that HpaXpm induced the hypersensitive response (HR), defense responses, and growth promotion in non-host plants more effectively than Hp1Xoo (X. oryzae pv. oryzae). Quantitative real-time PCR analysis indicated that HpaXpm proteins subjected to heat treatments at 100 °C, 150 °C, or 200 °C were still able to stimulate the expression of function-related genes (i.e., the HR marker genes Hin1 and Hsr203J, the defense-related gene NPR1, and the plant growth enhancement-related gene NtEXP6); however, the ability of heat-treated HpaXpm to induce HR was different at different temperatures. Conclusions These findings add a new member to the harpin family. HpaXpm is heat-stable up to 200 °C and is able to stimulate powerful beneficial biological functions that could potentially be more valuable for agricultural applications than those stimulated by Hpa1Xoo. We hypothesize that the extreme heat resistance of HpaXpm is because the structure of harpin is very stable and, therefore, the HpaXpm structure is less affected by temperature.

Although conventionally considered an animal pathogen, recent evidence increasingly suggests that fresh produce may act as significant transmission vehicles and alternative hosts to Salmonella. This study reports the differential survivability of two genetically similar Salmonella Thompson strains (ST 889B and ST 688C) on the adaxial surface of pre-harvest basil (Ocimum basilicum) leaves. Upon inoculation, two distinct phenomena, a dried water-print or a macroscopic lesion, were observed within 24 h. ST 889B survived better than ST 688C on healthy-looking leaves without lesions, possibly due to its higher biofilm-forming ability. Both strains survived better on the leaves with lesions than on the healthy-looking leaves (ST 688C: 4.39 ± 0.68 vs. 2.18 ± 0.29; ST 889B: 4.78 ± 0.12 vs. 2.83 ± 0.18 log CFU per sample at 6 days post-inoculation). ST 889B caused the formation of lesions at a higher frequency [70/117 leaves (59.8%)] than ST 688C [35/96 leaves (36.5%)]. Thus, we highlighted two distinct Salmonella survival strategies in the basil pathosystem and demonstrated gene expression polymorphism (variations in the expression of the same set of genes) as an indispensable strategy in the colonization of plants as hosts by the human pathogens.

Effector-triggered immunity (ETI) is one of the most studied mechanisms of plant resistance to viruses. During ETI, viral proteins are recognized by specific plant R proteins, which most often trigger a hypersensitive response (HR) involving programmed cell death (PCD) and a restriction of infection in the initially infected sites. However, in some plant–virus interactions, ETI leads to a response in which PCD and virus multiplication are not restricted to the entry sites and spread throughout the plant, leading to systemic necrosis. The host and virus genetic determinants, and the consequences of this response in plant–virus coevolution, are still poorly understood. Here, we identified an allelic version of RCY1—an R protein—as the host genetic determinant of broad-spectrum systemic necrosis induced by cucumber mosaic virus (CMV) infection in the Arabidopsis thaliana Co-1 ecotype. Systemic necrosis reduced virus fitness by shortening the infectious period and limiting virus multiplication; thus, this phenotype could be adaptive for the plant population as a defense against CMV. However, the low frequency (less than 1%) of this phenotype in A. thaliana wild populations argues against this hypothesis. These results expand current knowledge on the resistance mechanisms to virus infections associated with ETI in plants.

Abstract Here, we report a novel protein elicitor from Bacillus subtilis BU412 which could cause hypersensitive response (HR) and systemic acquired resistance (SAR) in tobacco. The purification was executed by ion-exchange and size exclusion chromatography. The target band on SDS-PAGE was analyzed by mass spectrometry, and the peptide mass fingerprinting matched an uncharacterized protein (WP_017418614.1), which was then named AMEP412. AMEP412 could cause a clearly defined HR necrosis in tobacco leaves, which was less affected by thermal treatment. The sub-cellular localization assay revealed that AMEP412 localized on the cell surface. This protein could also trigger early defense events such as the generation of reactive oxygen species (H2O2 and O2 −) and the induction of defense enzymes, including superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL). Moreover, AMEP412 could stimulate plant systemic resistance against Pseudomonas syringae pv. tomato DC3000.

Ceratocystis manginecans causes mango wilt with significant economic losses. In the infection court, cerato-platanin (CP) family proteins (CPPs) are believed to involve in pathogenesis but has not been determined in C. manginecans. To confirm this function, a CP protein (CmCP) of C. manginecans was characterized in this study. A protoplast of C. manginecans was prepared by treating its mycelia with driselase and lysing enzymes. The cmcp gene was edited using CRISPR/Cas-U6-1 expression vectors in 60% PEG and 50 μg/mL hygromycin B in the medium, resulting in mutants with cmcp deletion (Δcmcp). A complemented mutant (Δcmcp-C) was obtained by transforming cmcp to Δcmcp. Both Δcmcp and Δcmcp-C were characterized by comparing them with a wild-type strain on morphology, mycelial growth, conidial production and pathogenicity. Additionally, cmcp was transformed and expressed in Pichia pastoris, and the derived recombinant protein CmCP caused a severe necrosis on Nicotiana tabacum leaves. CmCP-treated plant leaves showed symptoms of hypersensitive response including electrolyte leakage, reactive oxygen species generation and overexpression of defense-related genes PR-1, PAD3, ERF1, HSR203J, and HIN1. All those results suggested that cmcp gene was required for the growth development of C. manginecans and functioned as a major pathogenicity factor in mango infection.

Citrus leprosis virus C (CiLV-C) belongs to the genus Cilevirus, family Kitaviridae, and is considered the most devastating virus infecting citrus in Brazil, being the main viral pathogen responsible for citrus leprosis (CL), a severe disease that affects citrus orchards in Latin America. Here, proteins encoded by CiLV-C genomic RNA 1 and 2 were screened for potential RNA silencing suppressor (RSS) activity by five methods. Using the GFP-based reporter agroinfiltration assay, we have not found potential local suppressor activity for the five CiLV-C encoded proteins. However, when RSS activity was evaluated using the alfalfa mosaic virus (AMV) system, we found that the p29, p15, and p61 CiLV-C proteins triggered necrosis response and increased the AMV RNA 3 accumulation, suggesting a suppressive functionality. From the analysis of small interfering RNAs (siRNAs) accumulation, we observed that the ectopic expression of the p29, p15, and p61 reduced significantly the accumulation of GFP derived siRNAs. The use of the RSS defective turnip crinkle virus (TCV) system revealed that only the trans-expression of the p15 protein restored the cell-to-cell viral movement. Finally, the potato virus X (PVX) system revealed that the expression of p29, p15, and p61 increased the PVX RNA accumulation; in addition, the p29 and p15 enhanced the pathogenicity of PVX resulting in the death of tobacco plants. Furthermore, PVX-p61 infection resulted in a hypersensitive response (HR), suggesting that p61 could also activate a plant defense response mechanism. This is the first report describing the RSS activity for CiLV-C proteins and, moreover, for a member of the family Kitaviridae.

Virus infection of plants can result in various degrees of detrimental impacts and disparate symptom types and severities. Although great strides have been made in our understanding of the virus–host interactions in herbaceous model plants, the mechanisms underlying symptom development are poorly understood in perennial fruit crops. Grapevine fanleaf virus (GFLV) causes variable symptoms in most vineyards worldwide. To better understand GFLV-grapevine interactions in relation to symptom development, field and greenhouse trials were conducted with a grapevine genotype that exhibits distinct symptoms in response to a severe and a mild strain of GFLV. After validation of the infection status of the experimental vines by high-throughput sequencing, the transcriptomic and metabolomic profiles in plants infected with the two viral strains were tested and compared by RNA-Seq and LC-MS, respectively, in the differentiating grapevine genotype. In vines infected with the severe GFLV strain, 1023 genes, among which some are implicated in the regulation of the hypersensitive-type response, were specifically deregulated, and a higher accumulation of resveratrol and phytohormones was observed. Interestingly, some experimental vines restricted the virus to the rootstock and remained symptomless. Our results suggest that GFLV induces a strain- and cultivar-specific defense reaction similar to a hypersensitive reaction. This type of defense leads to a severe stunting phenotype in some grapevines, whereas others are resistant. This work is the first evidence of a hypersensitive-like reaction in grapevine during virus infection.

When Arabidopsis thaliana ecotype Col-0 was inoculated with a series of reassortant viruses created by exchanging viral genomic RNAs between two strains of cucumber mosaic virus (CMV), CMV(Y), and CMV(H), cell death developed in the leaves inoculated with reassortant CMV carrying CMV(H) RNA1 encoding 1a protein, but not in noninoculated upper leaves. In general, cell death in virus-infected plants is a critical event for virus survival because virus multiplication is completely dependent on host cell metabolism. However, interestingly, this observed cell death did not affect either virus multiplication in the inoculated leaves or systemic spread to noninoculated upper leaves. Furthermore, the global gene expression pattern of the reassortant CMV-inoculated leaves undergoing cell death was clearly different from that in hypersensitive response (HR) cell death, which is coupled with resistance to CMV. These results indicated that the observed cell death does not appear to be HR cell death but rather necrotic cell death unrelated to CMV resistance. Interestingly, induction of this necrotic cell death depended on single amino acid substitutions in the N-terminal region surrounding the methyltransferase domain of the 1a protein. Thus, development of necrotic cell death might not be induced by non-specific damage as a result of virus multiplication, but by a virus protein-associated mechanism. The finding of CMV 1a protein-mediated induction of necrotic cell death in A. thaliana, which is not associated with virus resistance and HR cell death, has the potential to provide a new pathosystem to study the role of cell death in virus−host plant interactions.

The plant cell wall acts not only as a physical barrier, but also as a complex and dynamic structure that actively changes under different biotic and abiotic stress conditions. The question is, how are the different cell wall compounds modified during different interactions with exogenous stimuli such as pathogens? Plants exposed to viral pathogens respond to unfavorable conditions on multiple levels. One challenge that plants face under viral stress is the number of processes required for differential cell wall remodeling. The key players in these conditions are the cell wall genes and proteins, which can be regulated in specific ways during the interactions and have direct influences on the rebuilding of the cell wall structure. The cell wall modifications occurring in plants during viral infection remain poorly described. Therefore, this study focuses on cell wall dynamics as an effect of incompatible interactions between the potato virus Y (PVYNTN) and resistant potatoes (hypersensitive plant), as well as compatible (susceptible plant) interactions. Our analysis describes, for the first time, the expression of the potato expansin A3 (StEXPA3) and potato extensin 4 (StEXT4) genes in PVYNTN-susceptible and -resistant potato plant interactions. The results indicated a statistically significant induction of the StEXPA3 gene during a susceptible response. By contrast, we demonstrated the predominantly gradual activation of the StEXT4 gene during the hypersensitive response to PVYNTN inoculation. Moreover, the in situ distributions of expansins (StEXPAs), which are essential cell wall-associated proteins, and the hydroxyproline-rich glycoprotein (HRGP) extensin were investigated in two types of interactions. Furthermore, cell wall loosening was accompanied by an increase in StEXPA deposition in a PVYNTN-susceptible potato, whereas the HRGP content dynamically increased during the hypersensitive response, when the cell wall was reinforced. Ultrastructural localization and quantification revealed that the HRGP extensin was preferably located in the apoplast, but deposition in the symplast was also observed in resistant plants. Interestingly, during the hypersensitive response, StEXPA proteins were mainly located in the symplast area, in contrast to the susceptible potato where StEXPA proteins were mainly observed in the cell wall. These findings revealed that changes in the intracellular distribution and abundance of StEXPAs and HRGPs can be differentially regulated, depending on different types of PVYNTN−potato plant interactions, and confirmed the involvement of apoplast and symplast activation as a defense response mechanism.

Polyhydroxyalkanoates (PHAs) are intracellular carbon and energy storage materials produced in various microorganisms under nutrient-limited conditions. PhaR is a regulatory protein involved in PHA synthesis. Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important bacterial pathogens in rice and has PHA biosynthesis genes in its genome, but the biological function of phaR in Xoo is unknown. In this study, we investigated the effects of the mutagenesis of phaR gene in Xoo strain PXO99A. Compared to the wildtype, the PhaR gene knock-out mutant PXO99ΔphaR was hypermotile and showed decreased growth rates in both rich and limited nutrient media. PXO99ΔphaR also showed almost 75% decrease in extracellular polysaccharide (EPS) production. When inoculated in rice leaves by leaf-clipping method, PXO99ΔphaR displayed reduced virulence in terms of lesion length and bacterial multiplication compared with the wildtype strain. PXO99ΔphaR also showed enhanced hypersensitive response (HR) induction in the leaves of non-host Nicotiana benthamiana with elevated hpa1 gene expression. Introduction of a cosmid containing the phaR coding sequence restored the phenotypes of the mutant to those of the wildtype strain. These results suggest that PhaR gene is an important gene that affects multiple bacterial characteristics, including EPS production, growth rate, defense response induced harpin production and motility, related to its virulence in plant.

The black rot pathogen Xanthomonas campestris pv. campestris (Xcc) is a model organism for the study of plant bacterial pathogenesis mechanisms. In bacteria, σ factors serve as important regulatory elements that respond to various environmental signals and cues. Though Xcc encodes 15 putative σ factors little is known about their roles. As an approach to identify the potential role of each σ factor, we constructed mutations in each of the σ-factor genes as well as generating mutants deficient in multiple σ factors to assess these regulators potential additive functions. The work identified two σ70 factors essential for growth. Furthermore, the work discovered a third σ70 factor, RpoE1, important for virulence. Further studies revealed that RpoE1 positively regulates the expression of the hrp gene cluster that encodes the type III secretion system (T3SS) which determines the pathogenicity and hypersensitive response of Xcc on plants. In vivo and in vitro studies demonstrated that RpoE1 could bind to the promoter region and promote transcription of hrpX, a gene encoding a key regulator of the hrp genes. Overall, this systematic analysis reveals important roles in Xcc survival and virulence for previously uncharacterized σ70 factors that may become important targets for disease control.

Acidovorax citrulli causes bacterial fruit blotch, a disease that poses a global threat to watermelon and melon production. Despite its economic importance, relatively little is known about the molecular mechanisms of pathogenicity and virulence of A. citrulli. Like other plant-pathogenic bacteria, A. citrulli relies on a type III secretion system (T3SS) for pathogenicity. On the basis of sequence and operon arrangement analyses, A. citrulli was found to have a class II hrp gene cluster similar to those of Xanthomonas and Ralstonia spp. In the class II hrp cluster, hrpG and hrpX play key roles in the regulation of T3SS effectors. However, little is known about the regulation of the T3SS in A. citrulli. This study aimed to investigate the roles of hrpG and hrpX in A. citrulli pathogenicity. We found that hrpG or hrpX deletion mutants of the A. citrulli group II strain Aac5 had reduced pathogenicity on watermelon seedlings, failed to induce a hypersensitive response in tobacco, and elicited higher levels of reactive oxygen species in Nicotiana benthamiana than the wild-type strain. Additionally, we demonstrated that HrpG activates HrpX in A. citrulli. Moreover, transcription and translation of the type 3-secreted effector (T3E) gene Aac5_2166 were suppressed in hrpG and hrpX mutants. Notably, hrpG and hrpX appeared to modulate biofilm formation. These results suggest that hrpG and hrpX are essential for pathogenicity, regulation of T3Es, and biofilm formation in A. citrulli.

In this paper we have characterized the lineage of two traits associated with the coat proteins (CPs) of the tombusvirids: Silencing suppression and HR elicitation in Nicotiana species. We considered that the tombusvirid CPs might collectively be considered an effector, with the CP of each CP-encoding species comprising a structural variant within the family. Thus, a phylogenetic analysis of the CP could provide insight into the evolution of a pathogen effector. The phylogeny of the CP of tombusvirids indicated that CP representatives of the family could be divided into four clades. In two separate clades the CP triggered a hypersensitive response (HR) in Nicotiana species of section Alatae but did not have silencing suppressor activity. In a third clade the CP had a silencing suppressor activity but did not have the capacity to trigger HR in Nicotiana species. In the fourth clade, the CP did not carry either function. Our analysis illustrates how structural changes that likely occurred in the CP effector of progenitors of the current genera led to either silencing suppressor activity, HR elicitation in select Nicotiana species, or neither trait.

Tomato spotted wilt virus (TSWV) is one of the most destructive viral pathogens of plants. Recently, a single dominant gene conferring complete resistance to TSWV (RTSW) was identified in Nicotina alata and introgressed into cultivated tobacco (N. tabacum). However, whether the TSWV carries an avirulence (Avr) factor directed against RTSW remains obscure. In the present study, we identified the non-structural protein (NSm), the movement protein of TSWV, which is an RTSW-specific Avr factor, by using two different transient expression systems. Using amino acid (aa) substitution mutants, we demonstrated the ability to induce RTSW-mediated hypersensitive response (HR) of NSm is independent of its movement function. Moreover, key substitutions (C118Y and T120N), a 21-aa viral effector epitope, and different truncated versions of NSm, which are responsible for the recognition of the Sw-5b resistance gene of tomato, were tested for their ability to trigger HR to TSWV in tobacco. Together, our results demonstrated that RTSW-mediated resistance is triggered by NSm in the same way as by Sw-5b, however, via different elicitor active sites. Finally, an Avr gene-based diagnostic approach was established and used to determine the presence and effectiveness of resistance genes in tobacco.

The HrpN (harpin) protein of the fire blight pathogen Erwinia amylovora is an essential virulence factor secreted via the bacterial type III secretion system. HrpN also has avirulence activity when delivered to tobacco by E. amylovora and has defense elicitor activity when applied to plants as a cell-free protein extract. Here, we characterize a series of random mutations in hrpN that altered the predicted amino acid sequence of the protein. Amino acid substitutions and deletions in the highly conserved, C-terminal portion of HrpN disrupted the virulence and avirulence activities of the protein. Several of these mutations produced a dominant-negative effect on E. amylovora avirulence on tobacco. None of the mutations clearly separated the virulence and avirulence activities of HrpN. Some C-terminal mutations abolished secretion of HrpN by E. amylovora. The results indicate that the C-terminal half of HrpN is essential for its secretion by E. amylovora, for its virulence activity on apple and pear, and for its avirulence activity on tobacco. In contrast, the C-terminal half of HrpN was not required for cell-free elicitor activity. This suggests that the N-terminal and C-terminal halves of HrpN mediate cell-free elicitor activity and avirulence activity, respectively.

Rx-mediated resistance was analyzed in Rx-expressing transgenic Nicotiana plants. The infection outcome of nine Potato virus X isolates mutated at amino acid positions 121 and 127 of the coat protein (CP) confirmed the key role of these amino acids but provided a more complex picture than previously reported. In particular, in Rx-expressing Nicotiana spp., eliciting activity modulated by amino acid 121 was conditioned by the nature of amino acid 127. These results suggest that the specificity of recognition might be modulated by host factors that are somehow subtly modified between Rx-expressing potato and Rx-expressing transgenic Nicotiana plants. Moreover, the CP of three Potexviruses, Narcissus mosaic virus (NMV), White clover mosaic virus (WClMV), and Cymbidium mosaic virus (CymMV), are all recognized by the Rx-based machinery and able to trigger an Rx-dependant hypersensitive response. A smaller elicitor of 90 amino acids was identified in the CP of NMV and WClMV, which contains the previously identified key positions 121 and 127. This elicitor is only weakly conserved (approximately 40% identity) among the CP of the various recognized viruses, suggesting that the Rx molecular machinery targets a conserved structural element of the Potexvirus CP rather than a conserved amino acid motif.

The injection of nearly 30 effector proteins by the type III secretion system underlies the ability of Pseudomonas syringae pv. tomato DC3000 to cause disease in tomato and other host plants. The search for effector functions is complicated by redundancy within the repertoire and by plant resistance (R)-gene sentinels, which may convert effector virulence activities into a monolithic defense response. On the premise that some effectors target universal eukaryotic processes and that yeast (Saccharomyces cerevisiae) lacks R genes, the DC3000 effector repertoire was expressed in yeast. Of 27 effectors tested, HopAD1, HopAO1, HopD1, HopN1, and HopU1 were found to inhibit growth when expressed from a galactose-inducible GAL1 promoter, and HopAA1-1 and HopAM1 were found to cause cell death. Catalytic site mutations affecting the tyrosine phosphatase activity of HopAO1 and the cysteine protease activity of HopN1 prevented these effectors from inhibiting yeast growth. Expression of HopAA1-1, HopAM1, HopAD1, and HopAO1 impaired respiration in yeast, as indicated by tests with ethanol glycerol selective media. HopAA1-1 colocalized with porin to yeast mitochondria and was shown to cause cell death in yeast and plants in a domain-dependent manner. These results support the use of yeast for the study of plant-pathogen effector repertoires.

Effector proteins injected by the pathogenic bacteria Pseudomonas syringae into plants can have profound effects on the pathogen–host interaction due to their efficient recognition by plants and the subsequent triggering of defenses. The AvrRpt2 effector triggers strong local and systemic defense (called systemic acquired resistance [SAR]) responses in Arabidopsis thaliana plants that harbor a functional RPS2 gene that encodes an R protein in the coiled-coil, nucleotide-binding domain, leucine-rich repeat class. The newly identified win3-T mutant shows greatly reduced resistance to P. syringae carrying avrRpt2. In win3-T plants, RIN4 cleavage, an early AvrRpt2-induced event, is normal. However, salicylic acid accumulation is compromised, as is SAR induction and the local hypersensitive cell death response after infection by P. syringae carrying avrRpt2. WIN3 encodes a member of the firefly luciferase protein superfamily. Expression of WIN3 at an infection site partially requires PAD4, a protein known to play a quantitative role in RPS2-mediated signaling. WIN3 expression in tissue distal to an infection site requires multiple salicylic acid regulatory genes. Finally, win3-T plants show modestly increased susceptibility to virulent P. syringae and modestly reduced SAR in response to P. syringae carrying avrRpm1. Thus, WIN3 is a key element of the RPS2 defense response pathway and a basal and systemic defense component.

The biotrophic fungal pathogen Cladosporium fulvum (syn. Passalora fulva) is the causal agent of tomato leaf mold. The Avr4 protein belongs to a set of effectors that is secreted by C. fulvum during infection and is thought to play a role in pathogen virulence. Previous studies have shown that Avr4 binds to chitin present in fungal cell walls and that, through this binding, Avr4 can protect these cell walls against hydrolysis by plant chitinases. In this study, we demonstrate that Avr4 expression in Arabidopsis results in increased virulence of several fungal pathogens with exposed chitin in their cell walls, whereas the virulence of a bacterium and an oomycete remained unaltered. Heterologous expression of Avr4 in tomato increased the virulence of Fusarium oxysporum f. sp. lycopersici. Through tomato GeneChip analyses, we demonstrate that Avr4 expression in tomato results in the induced expression of only a few genes. Finally, we demonstrate that silencing of the Avr4 gene in C. fulvum decreases its virulence on tomato. This is the first report on the intrinsic function of a fungal avirulence protein that has a counter-defensive activity required for full virulence of the pathogen.

Pseudomonas aeruginosa 7NSK2 induces resistance in dicots through a synergistic interaction of the phenazine pyocyanin and the salicylic acid-derivative pyochelin. Root inoculation of the monocot model rice with 7NSK2 partially protected leaves against blast disease (Magnaporthe grisea) but failed to consistently reduce sheath blight (Rhizoctonia solani). Only mutations interfering with pyocyanin production led to a significant decrease in induced systemic resistance (ISR) to M. grisea, and in trans complementation for pyocyanin production restored the ability to elicit ISR. Intriguingly, pyocyanin-deficient mutants, unlike the wild type, triggered ISR against R. solani. Hence, bacterial pyocyanin plays a differential role in 7NSK2-mediated ISR in rice. Application of purified pyocyanin to hydroponically grown rice seedlings increased H2O2 levels locally on the root surface as well as a biphasic H2O2 generation pattern in distal leaves. Co-application of pyocyanin and the antioxidant sodium ascorbate alleviated the opposite effects of pyocyanin on rice blast and sheath blight development, suggesting that the differential effectiveness of pyocyanin with respect to 7NSK2-triggered ISR is mediated by transiently elevated H2O2 levels in planta. The cumulative results suggest that reactive oxygen species act as a double-edged sword in the interaction of rice with the hemibiotroph M. grisea and the necrotroph R. solani.

Resistance against the leaf mold fungus Cladosporium fulvum is mediated by the tomato Cf proteins which belong to the class of receptor-like proteins and indirectly recognize extracellular avirulence proteins (Avrs) of the fungus. Apart from triggering disease resistance, Avrs are believed to play a role in pathogenicity or virulence of C. fulvum. Here, we report on the avirulence protein Avr4, which is a chitin-binding lectin containing an invertebrate chitin-binding domain (CBM14). This domain is found in many eukaryotes, but has not yet been described in fungal or plant genomes. We found that interaction of Avr4 with chitin is specific, because it does not interact with other cell wall polysaccharides. Avr4 binds to chitin oligomers with a minimal length of three N-acetyl glucosamine residues. In vitro, Avr4 protects chitin against hydrolysis by plant chitinases. Avr4 also binds to chitin in cell walls of the fungi Trichoderma viride and Fusarium solani f. sp. phaseoli and protects these fungi against normally deleterious concentrations of plant chitinases. In situ fluorescence studies showed that Avr4 also binds to cell walls of C. fulvum during infection of tomato, where it most likely protects the fungus against tomato chitinases, suggesting that Avr4 is a counter-defensive virulence factor.