Induction Of Hypersensitive Response Research Articles

Functional Characterization of RNA Silencing Suppressor Encoded by Cotton Leafroll Dwarf Virus

The P0 proteins encoded by Cotton leafroll dwarf virus (CLRDV) serve as viral suppressors of RNA silencing (VSR). CLRDV P0 proteins share a VSR-associated F-box-like motif. This study investigated the role of specific amino acid (aa) residues within the F-box-like motif, elucidating their implications for VSR potency, pathogenicity, and intracellular localization. Analysis of various single aa substitution mutants within the F-box-like motif to understand their effect on P0 proteins’ VSR potency revealed that specific mutations significantly affect P0’s ability to suppress RNA silencing, emphasizing the crucial role of the F-box-like motif. Subcellular localization examinations indicated that the P0 proteins associate with the nucleus and endoplasmic reticulum, which may be linked to their VSR function. Also, the induction of hypersensitive response (HR)-like symptoms and the accumulation of reactive oxygen species by P0 proteins was observed, suggesting their role as pathogenicity factors. These results indicated that specific mutations significantly affect the functions of multifaceted P0 proteins, highlighting the F-box-like motif’s crucial role. Our study highlights the importance of further molecular investigations to elucidate how different CLRDV strains manifest various disease symptoms and severity. This is crucial considering the global economic importance of cotton and the potential emergence of more threatening CLRDV isolates.

A Putative Apoplastic Effector of Clavibacter capsici, ChpGCc as Hypersensitive Response and Virulence (Hrv) Protein in Plants.

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. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The diverse functions of Pseudomonas syringae syringae van Hall effectors in regulating the plant immune response

Sorghum relies on its immune system to defend against various pathogens, including Pseudomonas syringae syringae van Hall (Pss van Hall). However, the sorghum immune system is largely unknown. Reports on pathogenic effectors have provided valuable insights into the plant immune system; thus, we aimed to identify Pss van Hall effectors that can regulate the sorghum defense response in this study. Here, we first established the sorghum-Pss van Hall pathosystem and found that type III effectors played critical roles in the virulence of Pss van Hall to sorghum. To predict its effectors, the whole genome of Pss van Hall was sequenced, and 18 effector-coding genes were identified. Among them, five effectors belong to the core effectors of Pseudomonas syringae pathovars, and two may be monocot pathogen-specific effectors. Pss van Hall triggered the hypersensitive response (HR) in Nicotiana benthamiana. We found that the effectors of Pss van Hall can be divided into cell death inducers and immune repressors by examining their functions in HR induction and repression of PTI marker gene, ROS production, and pathogen growth. Finally, the roles of core effectors HopAJ2 and HopAN1, and specific effector HopAX1 were further confirmed in the sorghum-Pss van Hall pathosystem. Importantly, the functions of HopAN1 and HopAX1 in regulating plant immunity were reported for the first time. We believe that the identification of these effectors will facilitate the continued exploration of the sorghum immune system.

A putative multi-sensor hybrid histidine kinase, BarA Ac , inhibits the expression of the type III secretion system regulator HrpG in Acidovorax citrulli.

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, severely damages watermelon, melon, and other cucurbit crops worldwide. Although many virulence determinants have been identified in A. citrulli, including swimming motility, twitching motility, biofilm formation, and the type III secretion system (T3SS), research on their regulation is lacking. To study virulence regulation mechanisms, we found a putative histidine kinase BarA Ac that may be related to the T3SS regulator HrpG in A. citrulli. We deleted and characterized barAAc (Aave_2063) in A. citrulli Aac5 strain. Compared to the wild-type Aac5, virulence and early proliferation of barAAc mutant in host watermelon cotyledons were significantly increased, and induction of hypersensitive response in non-host tobacco was accelerated, while biofilm formation and swimming motility were significantly reduced. In addition, the transcriptomic analysis revealed that the expression of many T3SS-related genes was upregulated in the ΔbarAAc deletion mutant when cultured in KB medium. Meanwhile, the ΔbarAAc deletion mutant showed increased accumulation of the T3SS regulator HrpG in KB medium, which may account for the increased deployment of T3SS. This suggests that the putative histidine kinase BarA Ac is able to repress the T3SS expression by inhibiting HrpG in the KB medium, which appears to be important for rational energy allocation. In summary, our research provides further understanding of the regulatory network of A. citrulli virulence.

Three amino acid residues are required for the recognition of Ralstonia solanacearum RipTPS in Nicotiana tabacum.

Ralstonia solanacearum causes devastating diseases in a wide range of economically important crops. It secretes a large number of virulence factors, also known as effectors, to promote its infection, and some of them are recognized when the host plant contains corresponding resistance genes. In this study we showed that a type III effector RipTPS from the avirulent R. solanacearum strain GMI1000 (RipTPSG) specifically induced cell death in Nicotiana tabacum, but not in Nicotiana benthamiana, whereas the RipTPS homolog in the virulent strain CQPS-1 (RipTPSC) induced cell death in neither N. tabacum nor N. benthamiana. These results indicated that RipTPSG is recognized in N. tabacum. Expression of RipTPSG induced upregulation of hypersensitive response (HR) -related genes in N. tabacum. The virulence of CQPS-1 was reduced when RipTPSG was genetically introduced into CQPS-1, further confirming that RipTPSG functions as an avirulence determinant. Protein sequence alignment indicated that there are only three amino acid polymorphisms between RipTPSG and RipTPSC. Site-directed mutagenesis analyses confirmed that the three amino acid residues are jointly required for the recognition of RipTPSG in N. tabacum. Expression of either RipTPSG or RipTPSC suppressed flg22-triggered reactive oxygen species (ROS) burst in N. benthamiana, suggesting that RipTPS contributes to pathogen virulence. Mutating the conserved residues in RipTPS’s trehalose-phosphate synthase (TPS) domain did not block its HR induction and defense suppression activity, indicating that the TPS activity is not required for RipTPS’s avirulence and virulence function.

Beauty and the pathogens: A leaf-less control presents a better image of Cymbidium orchids defense strategy.

Biological control is a safe way of combating plant diseases using the living organisms. For the precise use of microbial biological control agents, the genetic information on the hypersensitive response (HR), and defense-related gene induction pathways of plants are necessary. Orchids are the most prominent stakeholders of floriculture industry, and owing to their long-awaited flowering pattern, disease control is imperative to allow healthy vegetative growth that spans more than 2 years in most of the orchids. We observed leaf-less flowering in three orchid species (Cymbidium ensifolium, C. goeringii and C. sinense). Using these materials as reference, we performed transcriptome profiling for healthy leaves from non-infected plants to identify genes specifically involved in plant-pathogen interaction pathway. For this pathway, a total of 253 differentially expressed genes (DEGs) were identified in C. ensifolium, 189 DEGs were identified in C. goeringii and 119 DEGs were found in C. sinense. These DEGs were mainly related to bacterial secretion systems, FLS2, CNGCs and EFR, regulating HR, stomatal closure and defense-related gene induction. FLS2 (LRR receptor-like serine/threonine kinase) contained the highest number of DEGs among three orchid species, followed by calmodulin. Highly upregulated gene sets were found in C. sinense as compared to other species. The great deal of DEGs, mainly the FLS2 and EFR families, related to defense and immunity responses can effectively direct the future of biological control of diseases for orchids.

An Apoplastic Effector Pat-1Cm of the Gram-Positive Bacterium Clavibacter michiganensis Acts as Both a Pathogenicity Factor and an Immunity Elicitor in Plants.

Clavibacter michiganensis, a Gram-positive plant-pathogenic bacterium, utilizes apoplastic effectors for disease development in host plants. Here, we determine the roles of Pat-1Cm (a putative serine protease) in pathogenicity and plant immunity. Pat-1Cm was found to be a genuine secreted protein, and the secreted mature form did not carry the first 33 amino acids predicted to be a signal peptide (SP). The pat-1Cm mutant impaired to cause wilting, but still caused canker symptom in tomato. Moreover, this mutant failed to trigger the hypersensitive response (HR) in a nonhost Nicotiana tabacum. Among orthologs and paralogs of pat-1Cm, only chp-7Cs from Clavibacter sepedonicus, a potato pathogen, successfully complemented pat-1Cm function in pathogenicity in tomato, whereas all failed to complement pat-1Cm function in HR induction in N. tabacum. Based on the structural prediction, Pat-1Cm carried a catalytic triad for putative serine protease, and alanine substitution of any amino acids in the triad abolished both pathogenicity and HR-inducing activities of Pat-1Cm in C. michiganensis. Ectopic expression of pat-1Cm with an SP from tobacco secreted protein triggered HR in N. tabacum, but not in tomato, whereas a catalytic triad mutant failed to induce HR. Inoculation of the pat-1Cm mutant mixed with the mutant of another apoplastic effector CelA (cellulase) caused severe wilting in tomato, indicating that these two apoplastic effectors can functionally cooperate in pathogenicity. Overall, these results indicate that Pat-1Cm is a distinct secreted protein carrying a functional catalytic triad for serine protease and this enzymatic activity might be critical for both pathogenicity and HR-eliciting activities of Pat-1Cm in plants.

Role of Trehalose Synthesis in Ralstonia syzygii subsp. indonesiensis PW1001 in Inducing Hypersensitive Response on Eggplant (Solanum melongena cv. Senryo-nigou).

Ralstonia syzygii subsp. indonesiensis (Rsi, former name: Ralstonia solanacearum phylotype IV) PW1001, a causal agent of potato wilt disease, induces hypersensitive response (HR) on its non-host eggplant (Solanum melongena cv. Senryo-nigou). The disaccharide trehalose is involved in abiotic and biotic stress tolerance in many organisms. We found that trehalose is required for eliciting HR on eggplant by plant pathogen Rsi PW1001. In R. solanacearum, it is known that the OtsA/ OtsB pathway is the dominant trehalose synthesis pathway, and otsA and otsB encode trehalose-6-phosphate (T6P) synthase and T6P phosphatase, respectively. We generated otsA and otsB mutant strains and found that these mutant strains reduced the bacterial trehalose concentration and HR induction on eggplant leaves compared to wild-type. Trehalose functions intracellularly in Rsi PW1001 because addition of exogenous trehalose did not affect the HR level and ion leakage. Requirement of trehalose in HR induction is not common in R. solanacearum species complex because mutation of otsA in Ralstonia pseudosolanacearum (former name: Ralstonia solanacearum phylotype I) RS1002 did not affect HR on the leaves of its non-host tobacco and wild eggplant Solanum torvum. Further, we also found that each otsA and otsB mutant had reduced ability to grow in a medium containing NaCl and sucrose, indicating that trehalose also has an important role in osmotic stress tolerance.

Plant death caused by inefficient induction of antiviral R-gene-mediated resistance may function as a suicidal population resistance mechanism

Land plant genomes carry tens to hundreds of Resistance (R) genes to combat pathogens. The induction of antiviral R-gene-mediated resistance often results in a hypersensitive response (HR), which is characterized by virus containment in the initially infected tissues and programmed cell death (PCD) of the infected cells. Alternatively, systemic HR (SHR) is sometimes observed in certain R gene–virus combinations, such that the virus systemically infects the plant and PCD induction follows the spread of infection, resulting in systemic plant death. SHR has been suggested to be the result of inefficient resistance induction; however, no quantitative comparison has been performed to support this hypothesis. In this study, we report that the average number of viral genomes that establish cell infection decreased by 28.7% and 12.7% upon HR induction by wild-type cucumber mosaic virus and SHR induction by a single-amino acid variant, respectively. These results suggest that a small decrease in the level of resistance induction can change an HR to an SHR. Although SHR appears to be a failure of resistance at the individual level, our simulations imply that suicidal individual death in SHR may function as an antiviral mechanism at the population level, by protecting neighboring uninfected kin plants.

The stability of the coiled-coil structure near to N-terminus influence the heat resistance of harpin proteins from Xanthomonas

BackgroundHeat 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.ResultsEach 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.ConclusionsThe 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.

Ferric Uptake Regulator (FurA) is Required for Acidovorax citrulli Virulence on Watermelon.

Acidovorax citrulli is the causal agent of bacterial fruit blotch, a serious threat to commercial watermelon and melon crop production worldwide. Ferric uptake regulator (Fur) is a global transcription factor that affects a number of virulence-related functions in phytopathogenic bacteria; however, the role of furA has not been determined for A. citrulli. Hence, we constructed an furA deletion mutant and a corresponding complement in the background of A. citrulli strain xlj12 to investigate the role of the gene in siderophore production, concentration of intracellular Fe2+, bacterial sensitivity to hydrogen peroxide, biofilm formation, swimming motility, hypersensitive response induction, and virulence on melon seedlings. The A. citrulli furA deletion mutant displayed increased siderophore production, intracellular Fe2+ concentration, and increased sensitivity to hydrogen peroxide. In contrast, biofilm formation, swimming motility, and virulence on melon seedlings were significantly reduced in the furA mutant. As expected, complementation of the furA deletion mutant restored all phenotypes to wild-type levels. In accordance with the phenotypic results, the expression levels of bfrA and bfrB that encode bacterioferritin, sodB that encodes iron/manganese superoxide dismutase, fliS that encodes a flagellar protein, hrcN that encodes the type III secretion system (T3SS) ATPase, and hrcC that encodes the T3SS outer membrane ring protein were significantly downregulated in the A. citrulli furA deletion mutant. In addition, the expression of feo-related genes and feoA and feoB was significantly upregulated in the furA mutant. Overall, these results indicated that, in A. citrulli, FurA contributes to the regulation of the iron balance system, and affects a variety of virulence-related traits.

Two lytic transglycosylases of Xanthomonas campestris pv. campestris associated with cell separation and type III secretion system, respectively.

The lytic transglycosylases (LTs) are important enzymes that degrade peptidoglycan of the bacterial cell wall and affect many biological functions. We present here that XC_0706 and XC_3001 are annotated as the LTs in Xanthomonas campestris pv. campestris. XC_0706 is associated with virulence and plays a pivotal role in cell division. Mutation on XC_3001 reduced hypersensitive response induction and the translocation of type III effector, but did not affect the function of the type II secretion system. Further studies showed that multiple LTs genes contribute to efficiency of the type III secretory system in X. campestris pv. campestris.

Recognition of lettuce downy mildew effector BLR38 in Lactuca serriola LS102 requires two unlinked loci.

SummaryPlant‐pathogenic oomycetes secrete effector proteins to suppress host immune responses. Resistance proteins may recognize effectors and activate immunity, which is often associated with a hypersensitive response (HR). Transient expression of effectors in plant germplasm and screening for HR has proven to be a powerful tool in the identification of new resistance genes. In this study, 14 effectors from the lettuce downy mildew Bremia lactucae race Bl:24 were screened for HR induction in over 150 lettuce accessions. Three effectors—BLN06, BLR38 and BLR40—were recognized in specific lettuce lines. The recognition of effector BLR38 in Lactuca serriola LS102 did not co‐segregate with resistance against race Bl:24, but was linked to resistance against multiple other B. lactucae races. Two unlinked loci are both required for effector recognition and are located near known major resistance clusters. Gene dosage affects the intensity of the BLR38‐triggered HR, but is of minor importance for disease resistance.

Two virulent sRNAs identified by genomic sequencing target the type III secretion system in rice bacterial blight pathogen

BackgroundSmall non-coding RNA (sRNA) short sequences regulate various biological processes in all organisms, including bacteria that are animal or plant pathogens. Virulent or pathogenicity-associated sRNAs have been increasingly elucidated in animal pathogens but little is known about similar category of sRNAs in plant-pathogenic bacteria. This is particularly true regarding rice bacterial blight pathogen Xanthomonas oryzae pathovar oryzae (Xoo) as studies on the virulent role of Xoo sRNAs is very limited at present.ResultsThe number and genomic distribution of sRNAs in Xoo were determined by bioinformatics analysis based on high throughput sequencing (sRNA-Seq) of the bacterial cultures from virulence-inducing and standard growth media, respectively. A total of 601 sRNAs were identified in the Xoo genome and ten virulent sRNA candidates were screened out based on significant differences of their expression levels between the culture conditions. In addition, trans3287 and trans3288 were also selected as candidates due to high expression levels in both media. The differential expression of 12 sRNAs evidenced by the sRNA-Seq data was confirmed by a convincing quantitative method. Based on genetic analysis of Xoo ΔsRNA mutants generated by deletion of the 12 single sRNAs, trans217 and trans3287 were characterized as virulent sRNAs. They are essential not only for the formation of bacterial blight in a susceptible rice variety Nipponbare but also for the induction of hypersensitive response (HR) in nonhost plant tobacco. Xoo Δtrans217 and Δtrans3287 mutants fail to induce bacterial blight in Nipponbare and also fail to induce the HR in tobacco, whereas, genetic complementation restores both mutants to the wild type in the virulent performance and HR induction. Similar effects of gene knockout and complementation were found in the expression of hrpG and hrpX genes, which encode regulatory proteins of the type III secretion system. Consistently, secretion of a type III effector, PthXo1, is blocked in Δtrans217 or Δtrans3287 bacterial cultures but retrieved by genetic complementation to both mutants.ConclusionsThe genetic analysis characterizes trans217 and trans3287 as pathogenicity-associated sRNAs essential for the bacterial virulence on the susceptible rice variety and for the HR elicitation in the nonhost plant. The molecular evidence suggests that both virulent sRNAs regulate the bacterial virulence by targeting the type III secretion system.

Survey of the response of 82 domestic landraces of Zea mays to cucumber mosaic virus (CMV) reveals geographical region‐related resistance to CMV in Japan

Zea mays has been historically imported to Japan via two independent geographical routes: into southern Japan by trading with Europe in the 16th century and into northern Japan by import from North America in the 19th century. Breeding to genetically improve on quality traits and high yields has led to the current domestic landraces in each region. In a survey of 82 domestic landraces, nine out of 38 landraces originating from southern Japan showed complete immunity to cucumber mosaic virus yellow strain (CMV(Y)) without the formation of necrotic local lesions (NLLs). In contrast, three out of 44 landraces originating from northern Japan developed NLLs, but revealed no systemic spread of the virus. Due to the absence of good documentation on NLL formation in Z. mays, the response of domestic landraces Aso‐1 and Aso‐3, originating from Ibaraki in northern Japan, to a challenge with CMV(Y) and CMV(Ma‐1) was further analysed. Aso‐3 only formed NLL in response to CMV(Y) but not to CMV(Ma‐1). Moreover, in CMV(Y)‐inoculated Aso‐3, virus spread was restricted to the primary infection site and the expression of defence‐related genes was up‐regulated, whereas Aso‐1 became systemically infected with either CMV(Y) or CMV(Ma‐1). The response of Aso‐3 to CMV(Y) was inherited as a single dominant trait. Together, these results pointed towards the induction of hypersensitive response (HR)‐mediated resistance to CMV(Y) in Aso‐3. Although HR‐mediated resistance to viruses has been studied mainly in dicots, the pathosystem CMV–Z. mays may provide a model to investigate HR‐mediated resistance to viruses in monocot plants.

HpaP, a novel regulatory protein with ATPase and phosphatase activity, contributes to full virulence in Xanthomonas campestris pv. campestris.

The ability of the bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc) to cause disease is dependent on the type III secretion system (T3SS). Proteins of the Xcc T3SS are encoded by hrp (hypersensitive response and pathogenicity) genes and whose expression is mainly controlled by the regulators HrpG and HrpX. Here, we describe the identification and characterization of a previously unknown regulatory protein (named HpaP), which plays important role in hrp gene expression and virulence in Xcc. Clean deletion of hpaP demonstrated reduced virulence and HR (hypersensitive response) induction of Xcc and alterations in cell motility and stress tolerance. Global transcriptome analyses revealed that most hrp genes were down regulated in the hpaP mutant, suggesting HpaP positively regulates hrp genes. GUS activity assays implied that HpaP regulates the expression of hrp genes via controlling the expression of hrpX. Biochemical analyses revealed that HpaP protein had both ATPase and phosphatase activity. While further site-directed mutagenesis of conserved residues in the PTP loop (a protein tyrosine phosphatase signature) of HpaP resulted in the loss of both phosphatase activity and regulatory activity in virulence and HR. Taken together, the findings identify a new regulatory protein that controls hrp gene expression and virulence in Xcc.

A plant effector-triggered immunity signaling sector is inhibited by pattern-triggered immunity.

Since signaling machineries for two modes of plant-induced immunity, pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), extensively overlap, PTI and ETI signaling likely interact. In an Arabidopsis quadruple mutant, in which four major sectors of the signaling network, jasmonate, ethylene, PAD4, and salicylate, are disabled, the hypersensitive response (HR) typical of ETI is abolished when the Pseudomonas syringae effector AvrRpt2 is bacterially delivered but is intact when AvrRpt2 is directly expressed in planta These observations led us to discovery of a network-buffered signaling mechanism that mediates HR signaling and is strongly inhibited by PTI signaling. We named this mechanism the ETI-Mediating and PTI-Inhibited Sector (EMPIS). The signaling kinetics of EMPIS explain apparently different plant genetic requirements for ETI triggered by different effectors without postulating different signaling machineries. The properties of EMPIS suggest that information about efficacy of the early immune response is fed back to the immune signaling network, modulating its activity and limiting the fitness cost of unnecessary immune responses.

RpoN2- and FliA-regulated fliTX is indispensible for flagellar motility and virulence in Xanthomonas oryzae pv. oryzae

BackgroundBacterial blight of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important crop diseases in the world. More insights into the mechanistic regulation of bacterial pathogenesis will help us identify novel molecular targets for developing effective disease control strategies. A large flagellar gene cluster is regulated under a three-tiered hierarchy by σ54 factor RpoN2 and its activator FleQ, and σ28 factor FliA. A hypothetical protein gene fliTX is located upstream of rpoN2, however, how it is regulated and how it is related to bacterial behaviors remain to be elucidated.ResultsSequence alignment analysis indicated that FliTX in Xoo is less well conserved compared with FliT proteins in Escherichia coli, Salmonella typhimurium, and Pseudomonas fluorescens. Co-transcription of fliTX with a cytosolic chaperone gene fliS and an atypical PilZ-domain gene flgZ in an operon was up-regulated by RpoN2/FleQ and FliA. Significantly shorter filament length and impaired swimming motility were observed in ∆fliTX compared with those in the wildtype strain. ∆fliTX also demonstrated reduced disease lesion length and in planta growth in rice, attenuated ability of induction of hypersensitive response (HR) in nonhost tobacco, and down-regulation of type III secretion system (T3SS)-related genes. In trans expression of fliTX gene in ∆fliTX restored these phenotypes to near wild-type levels.ConclusionsThis study demonstrates that RpoN2- and FliA-regulated fliTX is indispensible for flagellar motility and virulence and provides more insights into mechanistic regulation of T3SS expression in Xoo.

Characterization of the incompatible interaction between Erwinia tracheiphila and non-host tobacco (Nicotiana tabacum)

Bacterial wilt is one of the most destructive diseases affecting a wide range of crops in the Cucurbits family including muskmelon (Cucumis melo), cucumber (Cucumis sativus), and squash (Cucubita pepo). The disease is caused by Erwinia tracheiphila, a Gram-negative and xylem-inhabiting species of Erwinia, which pathogenic mechanism is poorly understood. Many Gram-negative phytobacteria induce hypersensitive response (HR) in non-host plants, an immunity reaction triggered by pathogen recognition. With some exemptions, Erwinia species—notably E. amylovora, the causative agent of fire blight of rosaceous crops, and the reclassified soft rot pathogens, Pectobacterium and Dickeya species (formerly E. carotovora and E. chrysanthemi)—have been known to elicit HR in tobacco. However, concerning its pathogenic mechanism, the elicitation of classic HR has not been reported for some less-studied Erwinia species including E. tracheiphila. We characterized the induction of HR by the bacterial wilt pathogen in tobacco (Nicotiana tabacum cultivar ‘Xanthi’) using visual and physiological methods. We surveyed 21 E. tracheiphila strains and found that all of them elicited programmed cell death. Three strains (HCa1-5, UnisCu1-1, and MISpSq) fluorescently labeled with GFP could be visualized in the infiltrated leaves. We aligned the sequences of their HR-inducing protein, harpin (HrpN), predicted the secondary structures, and located the position of putative HR elicitors. We discovered differences between Cucurbita and Cucumis strains and found a close association of E. tracheiphila HrpN with those of Pantoea sp., Erwinia piriflorinigrans, and Erwinia pyrifoliae. Pre-infiltration of tobacco leaves with a lower cell population prevented HR following a subsequent challenge at the same area with HR-inducing levels of inoculum. The selected strains induced leaf conductivity levels similar to the HR-inducing E. amylovora strain E9, and their populations in the leaves decreased days after infiltration. Our results indicate that E. tracheiphila induces a classic HR in tobacco just like other HR-inducing Erwinia species.

Analysis of Magnaporthe oryzae Genome Reveals a Fungal Effector, Which Is Able to Induce Resistance Response in Transgenic Rice Line Containing Resistance Gene, Pi54.

Rice blast caused by Magnaporthe oryzae is one of the most important diseases of rice. Pi54, a rice gene that imparts resistance to M. oryzae isolates prevalent in India, was already cloned but its avirulent counterpart in the pathogen was not known. After decoding the whole genome of an avirulent isolate of M. oryzae, we predicted 11440 protein coding genes and then identified four candidate effector proteins which are exclusively expressed in the infectious structure, appresoria. In silico protein modeling followed by interaction analysis between Pi54 protein model and selected four candidate effector proteins models revealed that Mo-01947_9 protein model encoded by a gene located at chromosome 4 of M. oryzae, interacted best at the Leucine Rich Repeat domain of Pi54 protein model. Yeast-two-hybrid analysis showed that Mo-01947_9 protein physically interacts with Pi54 protein. Nicotiana benthamiana leaf infiltration assay confirmed induction of hypersensitive response in the presence of Pi54 gene in a heterologous system. Genetic complementation test also proved that Mo-01947_9 protein induces avirulence response in the pathogen in presence of Pi54 gene. Here, we report identification and cloning of a new fungal effector gene which interacts with blast resistance gene Pi54 in rice.