Rice Bacterial Blight Pathogen Research Articles

Bacteriophage-mediated biosynthesis of MnO2NPs and MgONPs and their role in the protection of plants from bacterial pathogens.

Xanthomonas oryzae pv. oryzae (Xoo) is the plant pathogen of Bacterial Leaf Blight (BLB), which causes yield loss in rice. In this study, the lysate of Xoo bacteriophage X3 was used to mediate the bio-synthesis of MgO and MnO2. The physiochemical features of MgONPs and MnO2NPs were observed via Ultraviolet - Visible spectroscopy (UV-Vis), X-ray diffraction (XRD), Transmission/Scanning electron microscopy (TEM/SEM), Energy dispersive spectrum (EDS), and Fourier-transform infrared spectrum (FTIR). The impact of nanoparticles on plant growth and bacterial leaf blight disease were evaluated. Chlorophyll fluorescence was used to determine whether the nanoparticles application were toxic to the plants. An absorption peak of 215 and 230 nm for MgO and MnO2, respectively, confirmed nanoparticle formation via UV-Vis. The crystalline nature of the nanoparticles was detected by the analysis of XRD. Bacteriological tests indicated that MgONPs and MnO2NPs sized 12.5 and 9.8 nm, respectively, had strong in vitro antibacterial effects on rice bacterial blight pathogen, Xoo. MnO2NPs were found to have the most significant antagonist effect on nutrient agar plates, while MgONPs had the most significant impact on bacterial growth in nutrient broth and on cellular efflux. Furthermore, no toxicity to plants was observed for MgONPs and MnO2NPs, indeed, MgONPs at 200 μg/mL significantly increased the quantum efficiency of PSII photochemistry on the model plant, Arabidopsis, in light (ΦPSII) compared to other interactions. Additionally, significant suppression of BLB was noted in rice seedlings amended with the synthesized MgONPs and MnO2NPs. MnO2NPs showed promotion of plant growth in the presence of Xoo compared to MgONPs. An effective alternative for the biological production of MgONPs and MnO2NPs was reported, which serves as an effective substitute to control plant bacterial disease with no phytotoxic effect.

Bioinspired Green Synthesis of Silver Nanoparticles Using Three Plant Extracts and Their Antibacterial Activity against Rice Bacterial Leaf Blight Pathogen Xanthomonas oryzae pv. oryzae.

Rice bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is responsible for a significant reduction in rice production. Due to the small impact on the environment, biogenic nanomaterials are regarded as a new type of antibacterial agent. In this research, three colloids of silver nanoparticles (AgNPs) were synthesized with different biological materials such as Arctium lappa fruit, Solanum melongena leaves, and Taraxacum mongolicum leaves, and called Al-AgNPs, Sm-AgNPs and Tm-AgNPs, respectively. The appearance of brown colloids and the UV-Visible spectroscopy analysis proved the successful synthesis of the three colloids of AgNPs. Moreover, FTIR and XRD analysis revealed the formation of AgNPs structure. The SEM and TEM analysis indicated that the average diameters of the three synthesized spherical AgNPs were 20.18 nm, 21.00 nm, and 40.08 nm, respectively. The three botanical AgNPs had the strongest bacteriostatic against Xoo strain C2 at 20 μg/mL with the inhibition zone of 16.5 mm, 14.5 mm, and 12.4 mm, while bacterial numbers in a liquid broth (measured by OD600) decreased by 72.10%, 68.19%, and 65.60%, respectively. Results showed that the three AgNPs could inhibit biofilm formation and swarming motility of Xoo. The ultrastructural observation showed that Al-AgNPs adhered to the surface of bacteria and broke the bacteria. Overall, the three synthetic AgNPs could be used to inhibit the pathogen Xoo of rice bacterial leaf blight.

Targeted isolation of biocontrol agents from plants through phytopathogen co-culture and pathogen enrichment

In a long-term symbiotic relationship between plants and pathogens, plants have evolved to harbor beneficially endophytic microbiomes, thereby conferring them the ability to resist infection by pathogens. This prompted us to establish a phytopathogen-based co-culture platform for the targeted isolation of potential biocontrol agents from plants via specific pathogen enrichment. In this study, we investigated three different phytopathogenic systems, including kiwifruit, turfgrass, and rice, and their infectious bacterial and/or fungal pathogens. By using the developed mono- or co-enrichment platform, we efficiently isolated three antimicrobial agents, including Bacillus safensis ZK-1 against Pseudomonas syringae pv. actinidiae that causes kiwifruit canker, Pseudomonas alcaligenes ZK-2 against Clarireedia paspali that causes dollar spot disease in turfgrass, and Bacillus velezensis ZK-3 against rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae and rice blast fungus Magnaporthe oryzae. We believe that the phytopathogenic co-culture and pathogen enrichment platform developed here is versatile and effective for the isolation of potential biocontrol agents with specific or broad-spectrum antimicrobial activities from plants of interest in a targeted and large-scale manner.

Enterobacter asburiae and Pantoea ananatis Causing Rice Bacterial Blight in China.

Rice bacterial blight is a devastating bacterial disease threatening rice yield all over the world and Xanthomonas oryzae pv. oryzae is traditionally believed to be the pathogen. In recent years, we have received diseased rice samples with symptoms of blighted leaves from Sichuan and Guangdong provinces, China. Pathogen isolation and classification identified two different enterobacteria as the causal agents, namely Enterobacter asburiae and Pantoea ananatis. Among them, E. asburiae was isolated from samples of both provinces, and P. ananatis was only isolated from the Sichuan samples. Different from rice foot rot pathogen Dickeya zeae EC1 and rice bacterial blight pathogen X. oryzae pv. oryzae PXO99A, strains SC1, RG1, and SC7 produced rare cell wall degrading enzymes (CWDEs) but more extrapolysaccharides (EPS). E. asburiae strains SC1 and RG1 produced bacteriostatic substances while P. ananatis strain SC7 produced none. Pathogenicity tests indicated that all of them infected monocotyledonous rice and banana seedlings, but not dicotyledonous potato, radish, or cabbage. Moreover, strain RG1 was most virulent, while strains SC1 and SC7 were similarly virulent on rice leaves, even though strain SC1 propagated significantly faster in rice leaf tissues than strain SC7. This study firstly discovered E. asburiae as a new pathogen of rice bacterial blight, and in some cases, P. ananatis could be a companion pathogen. Analysis on production of virulence factors suggested that both pathogens probably employ a different mechanism to infect hosts other than using cell wall degrading enzymes to break through host cell walls.

PCR-mediated identification and characterization of rice bacterial blight pathogen (Xanthomonas oryzae pv. Oryzae) isolates collected from Sindh province of Pakistan

PCR-mediated identification and characterization of rice bacterial blight pathogen (Xanthomonas oryzae pv. Oryzae) isolates collected from Sindh province of Pakistan

OsPG1 Encodes a Polygalacturonase that Determines Cell Wall Architecture and Affects Resistance to Bacterial Blight Pathogen in Rice

BackgroundPlant cell walls are the main physical barrier encountered by pathogens colonizing plant tissues. Alteration of cell wall integrity (CWI) can activate specific defenses by impairing proteins involved in cell wall biosynthesis, degradation and remodeling, or cell wall damage due to biotic or abiotic stress. Polygalacturonase (PG) depolymerize pectin by hydrolysis, thereby altering pectin composition and structures and activating cell wall defense. Although many studies of CWI have been reported, the mechanism of how PGs regulate cell wall immune response is not well understood.ResultsNecrosis appeared in leaf tips at the tillering stage, finally resulting in 3–5 cm of dark brown necrotic tissue. ltn-212 showed obvious cell death and accumulation of H2O2 in leaf tips. The defense responses were activated in ltn-212 to resist bacterial blight pathogen of rice. Map based cloning revealed that a single base substitution (G-A) in the first intron caused incorrect splicing of OsPG1, resulting in a necrotic phenotype. OsPG1 is constitutively expressed in all organs, and the wild-type phenotype was restored in complementation individuals and knockout of wild-type lines resulted in necrosis as in ltn-212. Transmission electron microscopy showed that thicknesses of cell walls were significantly reduced and cell size and shape were significantly diminished in ltn-212.ConclusionThese results demonstrate that OsPG1 encodes a PG in response to the leaf tip necrosis phenotype of ltn-212. Loss-of-function mutation of ltn-212 destroyed CWI, resulting in spontaneous cell death and an auto-activated defense response including reactive oxygen species (ROS) burst and pathogenesis-related (PR) gene expression, as well as enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo). These findings promote our understanding of the CWI mediated defense response.

An xa5 Resistance Gene-Breaking Indian Strain of the Rice Bacterial Blight Pathogen Xanthomonas oryzae pv. oryzae Is Nearly Identical to a Thai Strain.

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) constrains production in major rice growing countries of Asia. Xoo injects transcription activator-like effectors (TALEs) that bind to and activate host “susceptibility” (S) genes that are important for disease. The bacterial blight resistance gene xa5, which reduces TALE activity generally, has been widely deployed. However, strains defeating xa5 have been reported in India and recently also in Thailand. We completely sequenced and compared the genomes of one such strain from each country and examined the encoded TALEs. The two genomes are nearly identical, including the TALE genes, and belong to a previously identified, highly clonal lineage. Each strain harbors a TALE known to activate the major S gene SWEET11 strongly enough to be effective even when diminished by xa5. The findings suggest international migration of the xa5-compatible pathotype and highlight the utility of whole genome sequencing and TALE analysis for understanding and responding to breakdown of resistance.

Three Proteins (Hpa2, HrpF and XopN) Are Concomitant Type III Translocators in Bacterial Blight Pathogen of Rice.

Type III (T3) proteic effectors occupy most of the virulence determinants in eukaryote-pathogenic Gram-negative bacteria. During infection, bacteria may deploy a nanomachinery called translocon to deliver T3 effectors into host cells, wherein the effectors fulfill their pathological functions. T3 translocon is hypothetically assembled by bacterial translocators, which have been identified as one hydrophilic and two hydrophobic proteins in animal-pathogenic bacteria but remain unclear in plant pathogens. Now we characterize Hpa2, HrpF, and XopN proteins as concomitant T3 translocators in rice bacterial blight pathogen by analyzing pathological consequences of single, double, and triple gene knockout or genetic complementation. Based on these genetic analyses, Hpa2, HrpF, and XopN accordingly contribute to 46.9, 60.3, and 69.8% proportions of bacterial virulence on a susceptible rice variety. Virulence performances of Hpa2, HrpF, and XopN were attributed to their functions in essentially mediating from-bacteria-into-rice-cell translocation of PthXo1, the bacterial T3 effector characteristic of transcription factors targeting plant genes. On average, 61, 62, and 71% of PthXo1 translocation are provided correspondingly by Hpa2, HrpF, and XopN, while they cooperate to support PthXo1 translocation at a greater-than-95% extent. As a result, rice disease-susceptibility gene SWEET11, which is the regulatory target of PthXo1, is activated to confer bacterial virulence and induce the leaf blight disease in rice. Furthermore, the three translocators also undergo translocation, but only XopN is highly translocated to suppress rice defense responses, suggesting that different components of a T3 translocon deploy distinct virulence mechanisms in addition to the common function in mediating bacterial effector translocation.

Identification and characterization of a spotted-leaf mutant spl40 with enhanced bacterial blight resistance in rice

BackgroundSpotted leaf mutants show typical necrotic lesions that appear spontaneously in the absence of any pathogen attack. These mutants are often characterized to exhibit programmed cell death (PCD) and activation of plant defense responses resulting in enhanced disease resistance to multiple pathogens. Here, we reported a novel spotted-leaf mutant, spl40 that showed enhanced disease resistance response.ResultsInitially lesions appeared at leaf tips during seedling stage and gradually covered the whole leaf at the tillering stage. The lesion development was light-dependent. spl40 showed obvious cell death at and around the lesion, and burst of reactive oxygen species (ROS) was accompanied by disturbed ROS scavenging system. Photosynthetic capacity was compromised as evidenced by significant reductions in chlorophyll content, important photosynthesis parameters and downregulated expression of photosynthesis-related genes which ultimately led to poor performance of major agronomic traits. spl40 exhibited enhanced resistance to 14 out of 16 races of bacterial blight pathogen of rice, caused by Xanthomonas oryzae pv. oryzae, most probably though activation of SA and JA signaling pathways, owing to upregulated expression of SA and JA signaling genes, though the exact mechanism remain to be elucidated. The spotted-leaf phenotype was controlled by a novel single recessive nuclear gene. Genetic mapping combined with high throughput sequencing analysis identified Os05G0312000 as the most probable candidate gene. Sequencing of ORF revealed a single SNP change from C to T that resulted in non-synonymous change in amino acid residue from leucine to phenylalanine. Interestingly, the complementation plants did not display lesions before heading but showed lesions at the heading stage and the transgenic T1 progenies could be classified into 3 categories based on their lesion intensity, indicating the complex genetic nature of the spl40 mutation.ConclusionThe results obtained here clearly show that genes related to defense and PCD were upregulated in accordance with enhanced disease resistance and occurrence of PCD, whereas the photosynthetic capacity and overall ROS homeostasis was compromised in spl40. Our data suggest that a novel spotted-leaf mutant, spl40, would help to elucidate the mechanism behind lesion development involving programmed cell death and associated defense responses.

Phosphate solubilizers as antagonists for bacterial leaf blight with improved rice growth in phosphorus deficit soil

Multifaceted phosphate solubilizing bacteria (PSB) promote growth of plants by acting both as biofertilizer and biopesticide. The present study aimed to use native beneficial rhizobacteria to suppress rice Bacterial Leaf Blight (BLB) pathogen as well as to combat the plant in P deficiency without reduction in crop yield. In present investigation, of 50 PSB isolated from rice rhizosphere, three bacterial strains inhibited the BLB pathogen (18–25 mm), solubilized phosphorus (up to197 µg mL−1) produced IAA (1.7–14 µg mL−1). Gluconic acid produced by phosphate solubilizing antagonistic bacteria and molecular confirmation through amplification of glucose dehydrogenase genes (gcd) showed that gluconic acid may have dual role in P solubilization as well as BLB suppression. Plate germination assay showed that increase in vigor index (27%), root diameter (29%), root length volume-1 (57%) and root projection area (43%) were observed in inoculated seedlings as compared to uninoculated control. Plant inoculation studies under net house conditions showed BLB disease suppression (up to 56%) along with increase in plant P contents (up to 0.47% in straw and 1.26% in seed) in treatments inoculated with Pseudomonas spp. MR11, MR34 and Bacillus sp. MR42. Increase in the activity of defense related enzymes Phenylalanine ammonia lyase (PAL: 5.165 activity as µmole cinnamic acid g−1. f. wt.), Catalase (CAT: 2.88 activity g−1. f. wt.), Peroxidase (POD: 2.4 activity g−1. f. wt.), β, 1–3 glucanase (283 activity g−1. f. wt.) and Polyphenol oxidase (PPO: 65 activity g−1. f. wt.) were observed in response to inoculation of P-solubilizing antagonistic bacteria. BOX PCR finger printing and viable cell count coupled with Fluorescent in situ hybridization showed the competence and survival of inoculated bacteria in rhizosphere and phyllosphere. The findings of the current research work strongly encouraged the use of PSB strains for BLB suppression in phosphorus deficient soils.

A Strain of an Emerging Indian Xanthomonas oryzae pv. oryzae Pathotype Defeats the Rice Bacterial Blight Resistance Gene xa13 Without Inducing a Clade III SWEET Gene and Is Nearly Identical to a Recent Thai Isolate.

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) injects transcription activator-like effectors (TALEs) that bind and activate host “susceptibility” (S) genes important for disease. Clade III SWEET genes are major S genes for bacterial blight. The resistance genes xa5, which reduces TALE activity generally, and xa13, a SWEET11 allele not recognized by the cognate TALE, have been effectively deployed. However, strains that defeat both resistance genes individually were recently reported in India and Thailand. To gain insight into the mechanism(s), we completely sequenced the genome of one such strain from each country and examined the encoded TALEs. Strikingly, the two strains are clones, sharing nearly identical TALE repertoires, including a TALE known to activate SWEET11 strongly enough to be effective even when diminished by xa5. We next investigated SWEET gene induction by the Indian strain. The Indian strain induced no clade III SWEET in plants harboring xa13, indicating a pathogen adaptation that relieves dependence on these genes for susceptibility. The findings open a door to mechanistic understanding of the role SWEET genes play in susceptibility and illustrate the importance of complete genome sequence-based monitoring of Xoo populations in developing varieties with effective 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.

RNA-Seq analysis reveals insight into enhanced rice Xa7-mediated bacterial blight resistance at high temperature.

Plant disease is a major challenge to agriculture worldwide, and it is exacerbated by abiotic environmental factors. During some plant-pathogen interactions, heat stress allows pathogens to overcome host resistance, a phenomenon which could severely impact crop productivity considering the global warming trends associated with climate change. Despite the importance of this phenomenon, little is known about the underlying molecular mechanisms. To better understand host plant responses during simultaneous heat and pathogen stress, we conducted a transcriptomics experiment for rice plants (cultivar IRBB61) containing Xa7, a bacterial blight disease resistance (R) gene, that were infected with Xanthomonas oryzae, the bacterial blight pathogen of rice, during high temperature stress. Xa7-mediated resistance is unusual relative to resistance mediated by other R genes in that it functions better at high temperatures. Using RNA-Seq technology, we identified 8,499 differentially expressed genes as temperature responsive in rice cultivar IRBB61 experiencing susceptible and resistant interactions across three time points. Notably, genes in the plant hormone abscisic acid biosynthesis and response pathways were up-regulated by high temperature in both mock-treated plants and plants experiencing a susceptible interaction and were suppressed by high temperature in plants exhibiting Xa7-mediated resistance. Genes responsive to salicylic acid, an important plant hormone for disease resistance, were down-regulated by high temperature during both the susceptible and resistant interactions, suggesting that enhanced Xa7-mediated resistance at high temperature is not dependent on salicylic acid signaling. A DNA sequence motif similar to known abscisic acid-responsive cis-regulatory elements was identified in the promoter region upstream of genes up-regulated in susceptible but down-regulated in resistant interactions. The results of our study suggest that the plant hormone abscisic acid is an important node for cross-talk between plant transcriptional response pathways to high temperature stress and pathogen attack. Genes in this pathway represent an important focus for future study to determine how plants evolved to deal with simultaneous abiotic and biotic stresses.

OxyR-regulated catalase CatB promotes the virulence in rice via detoxifying hydrogen peroxide in Xanthomonas oryzae pv. oryzae.

BackgroundTo facilitate infection, Xanthomonas oryzae pv. oryzae (Xoo), the bacterial blight pathogen of rice, needs to degrade hydrogen peroxide (H2O2) generated by the host defense response via a mechanism that is mediated by the transcriptional regulator OxyR. The catalase (CAT) gene catB has previously been shown to belong to the OxyR regulon in Xoo. However, its expression patterns and function in H2O2 detoxification and bacterial pathogenicity on rice remain to be elucidated.ResultsThe catB gene encodes a putative catalase and is highly conserved in the sequenced strains of Xanthomonas spp. β-galactosidase analysis and electrophoretic mobility shift assays (EMSA) showed that OxyR positively regulated the transcription of catB by directly binding to its promoter region. The quantitative real-time PCR (qRT-PCR) assays revealed that the expression levels of catB and oxyR were significantly induced by H2O2. Deletion of catB or oxyR drastically impaired bacterial viability in the presence of extracellular H2O2 and reduced CAT activity, demonstrating that CatB and OxyR contribute to H2O2 detoxification in Xoo. In addition, ΔcatB and ΔoxyR displayed shorter bacterial blight lesions and reduced bacterial growth in rice compared to the wild-type stain, indicating that CatB and OxyR play essential roles in the virulence of Xoo.ConclusionsTranscription of catB is enhanced by OxyR in response to exogenous H2O2. CatB functions as an active catalase that is required for the full virulence of Xoo in rice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0887-0) contains supplementary material, which is available to authorized users.

Complete nucleotide sequence of a new filamentous phage, Xf109, which integrates its genome into the chromosomal DNA of Xanthomonas oryzae.

Unlike Ff-like coliphages, certain filamentous Inoviridae phages integrate their genomes into the host chromosome and enter a prophage state in their infectious cycle. This lysogenic life cycle was first reported for Xanthomonas citri Cf phage. However, except for the X. citri phages Cf and XacF1, complete genome sequence information about lysogenic Xanthomonas phages is not available to date. A proviral sequence of Xf109 phage was identified in the genome of Xanthomonas oryzae, the rice bacterial blight pathogen, and revived as infectious virions to lysogenize its host de novo. The genome of Xf109 phage is 7190 nucleotides in size and contains 12 predicted open reading frames in an organization similar to that of the Cf phage genome. Seven of the Xf109 proteins show significant sequence similarity to Cf and XacF1 phage proteins, while its ORF4 shares 92% identity with the major coat protein of X. phage oryzae Xf. Integration of Xf109 phage DNA into the host genome is site-specific, and the attP/attB sequence contains the dif core sequence 5'-TATACATTATGCGAA-3', which is identical to that of Cf, XacF1, and Xanthomonas campestris phage ϕLf. To my knowledge, this is the first complete genome sequence of a filamentous bacteriophage that infects X. oryzae.

A transcription activator-like effector from Xanthomonas oryzae pv. oryzicola elicits dose-dependent resistance in rice.

Xanthomonas spp. reduce crop yields and quality worldwide. During infection of their plant hosts, many strains secrete transcription activator-like (TAL) effectors, which enter the host cell nucleus and activate specific corresponding host genes at effector binding elements (EBEs) in the promoter. TAL effectors may contribute to disease by activating the expression of susceptibility genes or trigger resistance associated with the hypersensitive reaction (HR) by activating an executor resistance (R) gene. The rice bacterial leaf streak pathogen X. oryzae pv. oryzicola (Xoc) is known to suppress host resistance, and no host R gene has been identified against it, despite considerable effort. To further investigate Xoc suppression of host resistance, we conducted a screen of effectors from BLS256 and identified Tal2a as an HR elicitor in rice when delivered heterologously by a strain of the closely related rice bacterial blight pathogen X. oryzae pv. oryzae (Xoo) or by the soybean pathogen X. axonopodis pv. glycines. The HR required the Tal2a activation domain, suggesting an executor R gene. Tal2a activity was differentially distributed among geographically diverse Xoc isolates, being largely conserved among Asian isolates. We identified four genes induced by Tal2a in next-generation RNA sequencing experiments and confirmed them using quantitative real-time reverse transcription-polymerase chain reaction (qPCR). However, neither individual nor collective activation of these genes by designer TAL effectors resulted in HR. A tal2a knockout mutant of BLS256 showed virulence comparable with the wild-type, but plasmid-based overexpression of tal2a at different levels in the wild-type reduced virulence in a directly corresponding way. Overall, the results reveal that host resistance suppression by Xoc plays a critical role in pathogenesis. Further, the dose-dependent avirulence activity of Tal2a and the apparent lack of a single canonical target that accounts for HR point to a novel, activation domain-dependent mode of action, which might involve, for example, a non-coding gene or a specific pattern of activation across multiple targets.

The RpfB-Dependent Quorum Sensing Signal Turnover System Is Required for Adaptation and Virulence in Rice Bacterial Blight Pathogen Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, produces diffusible signal factor (DSF) family quorum sensing signals to regulate virulence. The biosynthesis and perception of DSF family signals require components of the rpf (regulation of pathogenicity factors) cluster. In this study, we report that RpfB plays an essential role in DSF family signal turnover in X. oryzae pv. oryzae PXO99A. The production of DSF family signals was boosted by deletion of the rpfB gene and was abolished by its overexpression. The RpfC/RpfG-mediated DSF signaling system negatively regulates rpfB expression via the global transcription regulator Clp, whose activity is reversible in the presence of cyclic diguanylate monophosphate. These findings indicate that the DSF family signal turnover system in PXO99A is generally consistent with that in Xanthomonas campestris pv. campestris. Moreover, this study has revealed several specific roles of RpfB in PXO99A. First, the rpfB deletion mutant produced high levels of DSF family signals but reduced extracellular polysaccharide production, extracellular amylase activity, and attenuated pathogenicity. Second, the rpfB/rpfC double-deletion mutant was partially deficient in xanthomonadin production. Taken together, the RpfB-dependent DSF family signal turnover system is a conserved and naturally presenting signal turnover system in Xanthomonas spp., which plays unique roles in X. oryzae pv. oryzae adaptation and pathogenesis.

Harpin Hpa1 Interacts with Aquaporin PIP1;4 to Promote the Substrate Transport and Photosynthesis in Arabidopsis.

Harpin proteins produced by plant-pathogenic Gram-negative bacteria are the venerable player in regulating bacterial virulence and inducing plant growth and defenses. A major gap in these effects is plant sensing linked to cellular responses, and plant sensor for harpin Hpa1 from rice bacterial blight pathogen points to plasma membrane intrinsic protein (PIP). Here we show that Arabidopsis AtPIP1;4 is a plasma membrane sensor of Hpa1 and plays a dual role in plasma membrane permeability of CO2 and H2O. In particular, AtPIP1;4 mediates CO2 transport with a substantial contribute to photosynthesis and further increases this function upon interacting with Hpa1 at the plasma membrane. As a result, leaf photosynthesis rates are increased and the plant growth is enhanced in contrast to the normal process without Hpa1-AtPIP1;4 interaction. Our findings demonstrate the first case that plant sensing of a bacterial harpin protein is connected with photosynthetic physiology to regulate plant growth.

Mutations in the Predicted Active Site of Xanthomonas oryzae pv. oryzae XopQ Differentially Affect Virulence, Suppression of Host Innate Immunity, and Induction of the HR in a Nonhost Plant.

Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, secretes a number of effectors through a type 3 secretion system. One of these effectors, called XopQ, is required for virulence and suppression of rice innate immune responses induced by the plant cell-wall-degrading enzyme lipase/esterase A (LipA). Bioinformatic analysis suggested that XopQ is homologous to inosine-uridine nucleoside hydrolases (NH). A structural model of XopQ with the protozoan Crithidia fasciculata purine NH suggested that D116 and Y279 are potential active site residues. X. oryzae pv. oryzae xopQ mutants (xopQ-/pHM1::xopQD116A and xopQ-/pHM1::xopQY279A) show reduced virulence on rice compared with xopQ-/pHM1::xopQ. The two predicted XopQ active site mutants (xopQ-/pHM1::xopQD116A and xopQ-/pHM1::xopQY279A) exhibit a reduced hypersensitive response (HR) on Nicotiana benthamiana, a nonhost. However, Arabidopsis lines expressing either xopQ or xopQY279A are equally proficient at suppression of LipA-induced callose deposition. Purified XopQ does not show NH activity on standard nucleoside substrates but exhibits ribose hydrolase activity on the nucleoside substrate analogue 4-nitrophenyl β-D-ribofuranoside. The D116A and Y279A mutations cause a reduction in biochemical activity. These results indicate that mutations in the predicted active site of XopQ affect virulence and induction of the HR but do not affect suppression of innate immunity.

Transgenic Expression of a Functional Fragment of Harpin Protein Hpa1 in Wheat Represses English Grain Aphid Infestation

The harpin protein Hpa1 produced by the rice bacterial blight pathogen promotes plant growth and induces plant resistance to pathogens and insect pests. The region of 10–42 residues (Hpa110–42) in the Hpa1 sequence is critical as the isolated Hpa110–42 fragment is 1.3-7.5-fold more effective than the full length in inducing plant growth and resistance. Here we report that transgenic expression of Hpa110–42 in wheat induces resistance to English grain aphid, a dominant species of wheat aphids. Hpa110–42-induced resistance is effective to inhibit the aphid behavior in plant preference at the initial colonization stage and repress aphid performances in the reproduction, nymph growth, and instar development on transgenic plants. The resistance characters are correlated with enhanced expression of defense-regulatory genes (EIN2, PP2-A, and GSL10) and consistent with induced expression of defense response genes (Hel, PDF1.2, PR-1b, and PR-2b). As a result, aphid infestations are alleviated in transgenic plants. The level of Hpa110–42-induced resistance in regard to repression of aphid infestations is equivalent to the effect of chemical control provided by an insecticide. These results suggested that the defensive role of Hpa110–42 can be integrated into breeding germplasm of the agriculturally significant crop with a great potential of the agricultural application.