Rice Bacterial Pathogen Research Articles

Horizontal transmission of a rice bacterial pathogen Pantoea ananatis among plants by rice planthoppers.

Pantoea ananatis is a bacterium commonly found in various agronomic crops and agricultural pests. In this study, we present findings on a genome-reduced strain of P. ananatis, known as Lstr, which was initially isolated from Laodelphax striatellus (small brown rice planthopper, SBPH). We identified Lstr as a plant pathogen causing disease in rice using Koch's postulates. The pathogenicity of Lstr on rice is comparable to that of Xanthomonas oryzae pv. oryzae, the main causative agent of rice bacterial blight. Through a series of experiments involving live insects, molecular investigations, and microscopy, we find that Lstr can accumulate within SBPH. Subsequently, Lstr can be transmitted from SBPH to rice plants, resulting in leaf blight, and can also be transmitted to other SBPH individuals. Collectively, our results suggest that SBPH serves as a vector for P. ananatis Lstr in rice plants. P. ananatis may encounter susceptible insect populations and become endemic through horizontal transmission from these insects. This could also be valuable for predicting future occurrences of bacterial leaf blight in rice and other crops caused by P. ananatis.

OsWRKY65 enhances immunity against fungal and bacterial pathogens in rice

Diverse bacterial and fungal pathogens attack plants, causing biotic stress and severe yield losses globally. These losses are expected to become more serious as climate change improves conditions for many pathogens. Therefore, identifying genes conferring broad-spectrum disease resistance and elucidating their underlying mechanisms provides important resources for plant breeding. WRKY transcription factors affect plant growth and stress responses. However, the functions of many WRKY proteins remain to be elucidated. Here, we demonstrated the role of rice (Oryza sativa) WRKY group III transcription factor OsWRKY65 in immunity. OsWRKY65 localized to the nucleus and acted as transcriptional repressor. Genetic and molecular functional analyses showed that OsWRKY65 increases resistance to the fungal pathogen Fusarium fujikuroi through downregulation of GA signaling and upregulation of JA signaling. Moreover, OsWRKY65 modulated the expression of the key genes that confer susceptibility or resistance to Xanthomonas oryzae pv. oryzae to enhance immunity against the pathogen. In particular, OsWRKY65 directly bound to the promoter region of OsSWEET13 and repressed its expression. Taken together, our findings demonstrate that the OsWRKY65 enhances resistance to fungal and bacterial pathogens in rice.

Photo‐Stimuli Smart Supramolecular Self‐Assembly of Azobenzene/β‐Cyclodextrin Inclusion Complex for Controlling Plant Bacterial Diseases

AbstractControllable and on‐demand delivery of supramolecular systems have received considerable attention in modern agricultural management, especially for managing intractable plant diseases. Here, an intelligent photoresponsive pesticide delivery system is reported based on β‐cyclodextrin (β‐CD) and azobenzene, which overcomes the resistance of phytopathogens caused by the irrational use of conventional pesticides. Antibacterial bioassays illustrated that designed azobenzene derivative 3a possesses the most efficient bioactivity with EC50 values of 0.52–25.31 µg mL−1 toward three typical phytopathogens. Moreover, the assembly of the supramolecular binary complex 3a@β‐CD is successfully elucidated and displays exceptional inhibitory activity on biofilm formation. Of note, this supramolecular binary complex significantly improves the water solubility, foliar surface wettability, and shows marked light‐responsive properties. In vivo anti‐Xoo assays reveal that 3a@β‐CD has excellent control efficiency (protective activity: 51.22%, curative activity: 48.37%) against rice bacterial blight pathogens, and their control efficiency can be elevated to values of 55.84% (protective activity) and 52.05% (curative activity) by UV–vis exposure. In addition, the 3a@β‐CD are non‐toxic toward various non‐target organisms. This study therefore offers new insights into the potential of host‐guest complexes as a feasible pesticide discovery strategy characterized by a safe, biocompatible, light‐responsive release, and antibiofilm properties for overcoming intractable plant bacterial diseases.

Novel broad-spectrum bacteriophages against Xanthomonas oryzae and their biocontrol potential in rice bacterial diseases.

Bacterial leaf blight (BLB) and bacterial leaf streak (BLS)-caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively-are two major bacterial diseases that threaten the safe production of rice, one of the most important food crops. Bacteriophages are considered potential biocontrol agents against rice bacterial pathogens, due to their host specificity and environmental safety. It is common for BLB and BLS to occur together in fields, which highlights the need for broad-spectrum phages capable of infecting both Xoo and Xoc. In this study, two lytic broad-spectrum phages (pXoo2106 and pXoo2107) that can infect various strains of Xoo and Xoc were assessed. Both phages belong to the class Caudoviricetes and one of them to the family Autographiviridae, while the other belongs to an unclassified family. Two phages alone or combined in a phage cocktail could effectively inhibit Xoo and Xoc growth in vitro. In an in vivo biocontrol experiment, the phage cocktail reduced the total CFU and significantly eased the symptoms caused by Xoo or Xoc. Our results suggest that pXoo2106 and pXoo2107 have a broad-spectrum host range targeting different X. oryzae strains, and have strong biocontrol potential in field applications against both BLB and BLS.

First report of rice bacterial leaf blight disease caused by Pantoea ananatis in the United States.

In August 2021, bacterial leaf blight-like symptoms were observed on 14 out of 570 rice genotypes (Oryza sativa) in research field plots of global rice germplasm grown in Arkansas (eXtra Figure S1. A & B). The disease was characterized by spreading lesions on leaves, panicle sterility and reduced yield in highly susceptible, mature rice germplasm. No spread of disease to nearby plants was observed. Isolations were performed at Colorado State University, where soakates from symptomatic leaves were spread onto nutrient agar. After 72 h at 28°C, uniform, distinct, yellow-colored bacterial colonies were observed. To screen for the presence of common rice bacterial pathogens, PCR amplification directly from colonies or from DNA isolated from symptomatic field-collected leaves was performed. Primers specific for Xanthomonas oryzae pvs. oryzae and oryzicola (Lang et al., 2010), Burkholderia glumae (Echeverri-Rico et al., 2021), and Pseudomonas fuscovaginae (Ash et al., 2014) did not amplify indicating these organisms were not present. Sequencing of 16S rRNA gene (Weisburg et al., 1991) amplicons suggested the bacteria belonged to the genera Pantoea and Sphingomonas (NCBI accession no. OP683332 and OP683333, respectively). Amplicons resulting from primers specific to the gyrB gene region of P. ananatis (Kini et al., 2021) were sequenced and the fragment was compared to the P. ananatis PA13 reference genome using a BLAST analysis. One candidate (AR358) showed 100% identity with the P. ananatis gyrB region. Primers specific for Sphingomonas sp. (Bangratz et al., 2020) confirmed the second candidate (AR359) as a Sphingomonas sp. The identity of P. ananatis was confirmed by the Plant Pathogen Confirmatory Diagnostics Laboratory (Beltsville, MD, USA). To determine pathogenicity, leaves from 7-day-old seedlings of rice (Oryza sativa) cultivar Kitaake were scissor-clip inoculated (Kauffman et al., 1973) with four different treatments and compared to control leaves inoculated with sterile water. Treatments for the experiment consisted of bacterial suspensions (108 CFU/ml) of the two candidate organisms, P. ananatis (strain AR358) or Sphingomonas sp. (strain AR359), individually or in a 1:1 ratio of P. ananatis:Sphingomonas sp., or soakate from infected field tissue. Lesions similar to those observed in the field were only detected on leaves inoculated with P. ananatis or infected field tissue soakate at 7-days post-inoculation (eXtra Figure S1. C). Bacteria were recovered from the leaves of the artificially inoculated seedlings from three treatments (P. ananatis, P. ananatis:Sphingomonas sp. and soakate from the infected field tissue) and were determined to be P. ananatis based on colony morphology, amplification of 16s rRNA, and gyrB sequence data. Our results confirm the pathogenicity of P. ananatis to rice and fulfill Koch's postulates. P. ananatis was also recovered from several similarly diseased rice breeding lines at the University of Arkansas System Division of Agriculture Rice Research and Extension Center. We conclude that P. ananatis is the causal pathogen for leaf blight-like symptoms observed in the global rice cultivars grown in Arkansas. P. ananatis was previously reported as a pathogen on rice in several rice growing regions, including China (Yu et al., 2021), India (Reshma et al., 2022), and Africa (Kini et al., 2017), however, this is the first report of P. ananatis as a pathogen of rice in the United States.

Characterization of the chemical fungicides-responsive and bacterial pathogen-preventing Bacillus licheniformis in rice spikelet

Abstract Seed-borne bacterial pathogens cause severe yield loss and biotoxin contamination in rice, leading to increasing concern on the global food supply and environmental safety. Plant native microbes play an important role in defending against diseases, but their actions are often influenced by the chemical fungicides applied in the field. Here, Bacillus licheniformis mmj was isolated from rice spikelet, which uniquely showed not only fungicide-responsiveness but also broad-spectrum antimicrobial activity against major rice bacterial pathogens including Xanthomonas oryzae pv. oryzae, Burkholderia plantarii and Burkholderia glumae. To understand the hallmark underlying the environmental adaptation and antimicrobial activity of B. licheniformis mmj, the genome sequence was determined by SMRT and subjected to bioinformatics analysis. Genome sequence analysis enabled the identification of a set of antimicrobial-resistance and antibacterial activity genes together with an array of harsh environment-adaptive genes. Moreover, B. licheniformis mmj metabolites were analyzed with gas chromatography coupled to triple quadrupole mass spectrometry, and the volatile components that were linked with the antimicrobial activity were preliminarily profiled. Collectively, the present findings reveal the genomic and metabolic landscapes underlying fungicide-responsive B. licheniformis, which offers a new opportunity to design harsh environment-adaptive biopesticides to cope with prevalent bacterial phytopathogens.

Advances in Durable Resistance to Diseases in Staple Food Crops: A Review

Background: At all stages of their development, plants are in permanent contact with causative agents of various diseases. Mechanisms of disease resistance and its durability in crops largely depend on the pathogen’s lifestyle, namely the nutrition mode and host range. Objective: The objective of this review is to consider the main advances in the production of genotypes with durable disease resistance in the globally important food crops, wheat, rice, and potato, as well as barley. Results: In wheat, durable resistance could be provided by the employment of various adult plant resistance genes against biotrophic pathogens, whose action commonly does not involve hypersensitivity response, as well as major quantitative genes, including mutants of susceptibility alleles, against necrotrophs via marker assisted selection (MAS). In barley, the most prominent example is the gene mlo conferring durable powdery mildew resistance, but it is compromised by higher susceptibility to some necrotrophic fungi. A few genes for broad-spectrum resistance against the rice blast and bacterial blight pathogens confirmed their effectiveness for decades, and they could be combined with effective R genes via MAS. Resistance to late blight of potato is mainly provided by R genes introgressed from wild potato species, which could be pyramided with quantitative trait loci. Genes for extreme resistance to potato viruses derived from related species provide durable and broad-spectrum resistance and could be effectively deployed in potato breeding using MAS. Silencing susceptibility genes by genome editing technologies is the most promising approach to produce plants with durable resistance to many pathogens in the crop species. Genetic transformation with genes for resistance-associated proteins or constructs providing silencing via RNA interference is an effective biotechnological method to generate plants with durable resistance against pathogens, especially viruses. Conclusion: Main advances in the production of crop plants with durable resistance are based on studies of molecular mechanisms of plant immunity and its special features for pathogens with different lifestyles via the use of biotechnological approaches such as MAS for pyramiding of monogenic quantitative resistance genes or qualitative R genes, changes in expression of certain genes associated with resistance, the introduction of transgenes, mutagenesis and genome editing aimed at silencing susceptibility genes.

Osa-miR160a confers broad-spectrum resistance to fungal and bacterial pathogens in rice.

Rice production is threatened by multiple pathogens. Breeding cultivars with broad-spectrum disease resistance is necessary to maintain and improve crop production. Previously we found that overexpression of miR160a enhanced rice blast disease resistance. However, it is unclear whether miR160a also regulates resistance against other pathogens, and what the downstream signaling pathways are. Here, we demonstrate that miR160a positively regulates broad-spectrum resistance against the causative agents of blast, leaf blight and sheath blight in rice. Mutations of miR160a-targeted Auxin Response Factors result in different alteration of resistance conferred by miR160a. miR160a enhances disease resistance partially by suppressing ARF8, as mutation of ARF8 in MIM160 background partially restores the compromised resistance resulting from MIM160. ARF8 protein binds directly to the promoter and suppresses the expression of WRKY45, which acts as a positive regulator of rice immunity. Mutation of WRKY45 compromises the enhanced blast resistance and bacterial leaf blight resistance conferred by arf8 mutant. Overall, our results reveal that a microRNA coordinates rice broad-spectrum disease resistance by suppressing multiple target genes that play different roles in disease resistance, and uncover a new regulatory pathway mediated by the miR160a-ARF8 module. These findings provide new resources to potentially improve disease resistance for breeding in rice.

Seed priming with calcium chloride enhances stress tolerance in rice seedlings

Calcium is a crucial second messenger in plant cells and contributes to plant resistance against biotic and abiotic stress. Plant defense priming with natural or synthetic compounds leads to quicker and stronger resistance responses. However, whether pretreatment of plant seeds with calcium could improve their resistance to stress remains poorly understood. In this study, we showed that rice seedlings grown from calcium chloride (CaCl2)-pretreated seeds displayed enhanced resistance to the rice blast fungus Magnaporthe oryzae and the rice bacterial pathogen Xanthomonas oryzae pv. Oryzae (Xoo). Seed priming with CaCl2 also led to enhanced rice tolerance to salt and cold. Furthermore, the reactive oxygen species (ROS) burst increased significantly upon immunity activation in the leaves of rice seedlings grown from CaCl2-pretreated seeds. Additionally, we analyzed the rice calmodulin-binding protein 60 (OsCBP60) family and found that there were 19 OsCBP60s in rice cultivar Zhonghua 11 (ZH11). The transcripts of several OsCBP60s were chitin- and M. oryzae-inducible, suggesting that they may contribute to rice resistance. Taken together, these data indicate that seed priming with CaCl2 can effectively enhance rice tolerance to multiple stresses, perhaps by boosting the burst of ROS, and OsCBP60 family members may also play an essential role in this process.

Monoterpene geraniol produced by rice terpene synthase 21 suppresses the expression of cell-division related genes in the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae

Plant volatile compounds such as monoterpenes and sesquiterpenes represent some of the classes of plant volatile compounds and they are involved in the plant defense responses. They are synthesized by terpene synthases (TPSs). The rice genome has reportedly more than 30 TPSs genes and several of them are regulated by a plant hormone, jasmonic acid (JA). In the present study, we analyzed a rice terpene synthase, OsTPS21, and investigated its potential antibacterial effects against Xanthomonas oryzae pv. oryzae (Xoo), which causes rice bacterial blight. OsTPS21 was localized in the chloroplast and produced a monoterpene geraniol as a monoterpene synthase. In addition, OsTPS21 did not exhibit any activity as a sesquiterpene synthase. The amount of geraniol in rice increased along with the upregulation of OsTPS21 after JA treatment. Geraniol had a negative effect on the growth of Xoo. The growth inhibition of Xoo by geraniol was caused by the suppression of expression of the cell-division-related genes of Xoo and not by damage to Xoo cell membranes. The results suggest that geraniol plays an important role in JA-induced resistance against Xoo and that it functions as an antibacterial compound in rice.

Identification of a small set of genes commonly regulated in rice roots in response to beneficial rhizobacteria.

Rhizosphere bacteria, whether phytopathogenic or phytobeneficial, are thought to be perceived by the plant as a threat. Plant Growth-Promoting Rhizobacteria (PGPR), such as many strains of the Azospirillum genus known as the main phytostimulator of cereals, cooperate with host plants and favorably affect their growth and health. An earlier study of rice root transcriptome, undertaken with two rice cultivars and two Azospirillum strains, revealed a strain-dependent response during the rice-Azospirillum association and showed that only a few genes, including some implicated in plant defense, were commonly regulated in all tested conditions. Here, a set of genes was selected from previous studies and their expression was monitored by qRT-PCR in rice roots inoculated with ten PGPR strains isolated from various plants and belonging to various genera (Azospirillum, Herbaspirillum, Paraburkholderia). A common expression pattern was highlighted for four genes that are proposed to be markers of the rice-PGPR interaction: two genes involved in diterpenoid phytoalexin biosynthesis (OsDXS3 and OsDTC2) and one coding for an uncharacterized protein (Os02g0582900) were significantly induced by PGPR whereas one defense-related gene encoding a pathogenesis-related protein (PR1b, Os01g0382000) was significantly repressed. Interestingly, exposure to a rice bacterial pathogen also triggered the expression of OsDXS3 while the expression of Os02g0582900 and PR1b was down-regulated, suggesting that these genes might play a key role in rice-bacteria interactions. Integration of these results with previous data led us to propose that the jasmonic acid signaling pathway might be triggered in rice roots upon inoculation with PGPR.

Adverse effects of adaptive mutation to survive static culture conditions on successful fitness of the rice pathogen Burkholderia glumae in a host.

Bacteria often possess relatively flexible genome structures and adaptive genetic variants that allow survival in unfavorable growth conditions. Bacterial survival tactics in disadvantageous microenvironments include mutations that are beneficial against threats in their niche. Here, we report that the aerobic rice bacterial pathogen Burkholderia glumae BGR1 changes a specific gene for improved survival in static culture conditions. Static culture triggered formation of colony variants with deletions or point mutations in the gene bspP (BGLU_RS28885), which putatively encodes a protein that contains PDC2, PAS-9, SpoIIE, and HATPase domains. The null mutant of bspP survived longer in static culture conditions and produced a higher level of bis-(3′-5′)-cyclic dimeric guanosine monophosphate than the wild type. Expression of the bacterial cellulose synthase regulator (bcsB) gene was upregulated in the mutant, consistent with the observation that the mutant formed pellicles faster than the wild type. Mature pellicle formation was observed in the bspP mutant before pellicle formation in wild-type BGR1. However, the population density of the bspP null mutant decreased substantially when grown in Luria–Bertani medium with vigorous agitation due to failure of oxalate-mediated detoxification of the alkaline environment. The bspP null mutant was less virulent and exhibited less effective colonization of rice plants than the wild type. All phenotypes caused by mutations in bspP were recovered to those of the wild type by genetic complementation. Thus, although wild-type B. glumae BGR1 prolonged viability by spontaneous mutation under static culture conditions, such genetic changes negatively affected colonization in rice plants. These results suggest that adaptive gene sacrifice of B. glumae to survive unfavorable growth conditions is not always desirable as it can adversely affect adaptability in the host.

Blast resistance gene Pi54 over-expressed in rice to understand its cellular and sub-cellular localization and response to different pathogens

Rice blast resistance gene, Pi54 provides broad-spectrum resistance against different strains of Magnaporthe oryzae. Understanding the cellular localization of Pi54 protein is an essential step towards deciphering its place of interaction with the cognate Avr-gene. In this study, we investigated the sub-cellular localization of Pi54 with Green Fluorescent Protein (GFP) as a molecular tag through transient and stable expression in onion epidermal cells (Allium cepa) and susceptible japonica cultivar rice Taipei 309 (TP309), respectively. Confocal microscopy based observations of the onion epidermal cells revealed nucleus and cytoplasm specific GFP signals. In the stable transformed rice plants, GFP signal was recorded in the stomata, upper epidermal cells, mesophyll cells, vascular bundle, and walls of bundle sheath and bulliform cells of leaf tissues. These observations were further confirmed by Immunocytochemical studies. Using GFP specific antibodies, it was found that there was sufficient aggregation of GFP::Pi54protein in the cytoplasm of the leaf mesophyll cells and periphery of the epidermal cells. Interestingly, the transgenic lines developed in this study could show a moderate level of resistance to Xanthomonas oryzae and Rhizoctonia solani, the causal agents of the rice bacterial blight and sheath blight diseases, respectively. This study is a first detailed report, which emphasizes the cellular and subcellular distribution of the broad spectrum blast resistance gene Pi54 in rice and the impact of its constitutive expression towards resistance against other fungal and bacterial pathogens of rice.

A common metabolomic signature is observed upon inoculation of rice roots with various rhizobacteria.

Plant growth-promoting rhizobacteria (PGPR), whose growth is stimulated by root exudates, are able to improve plant growth and health. Among those, bacteria of the genus Azospirillum were shown to affect root secondary metabolite content in rice and maize, sometimes without visible effects on root architecture. Transcriptomic studies also revealed that expression of several genes involved in stress and plant defense was affected, albeit with fewer genes when a strain was inoculated onto its original host cultivar. Here, we investigated, via a metabolic profiling approach, whether rice roots responded differently and with gradual intensity to various PGPR, isolated from rice or not. A common metabolomic signature of nine compounds was highlighted, with the reduced accumulation of three alkylresorcinols and increased accumulation of two hydroxycinnamic acid amides (HCAA), identified as N-p-coumaroylputrescine and N-feruloylputrescine. This was accompanied by the increased transcription of two genes involved in the N-feruloylputrescine biosynthetic pathway. Interestingly, exposure to a rice bacterial pathogen triggered a reduced accumulation of these HCAA in roots, a result contrasting with previous reports of increased HCAA content in leaves upon pathogen infection. Accumulation of HCAA, that are potential antimicrobial compounds, might be considered as a primary reaction of plant to bacterial perception.

The host basal transcription factor IIA subunits coordinate for facilitating infection of TALEs-carrying bacterial pathogens in rice

Many plant-pathogenic Xanthomonas rely largely on secreting virulence transcription activator-like effectors (TALEs) proteins into plant nucleus to activate host susceptibility genes to cause disease, the process is dependent on pathogen TALEs association with host plants basal transcription factor IIA small subunit TFIIAγ. TFIIAγ together with large subunit TFIIAαβ constitute as a key component of RNA polymerase II complex for transcriptome initiation. However, whether TFIIAαβ coordinates or competes with pathogen TALEs for interaction with TFIIAγ to activate transcript of TALEs-targeting genes is unclear. Here, we showed that TALE-carrying bacterial pathogens Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent for bacterial leaf blight and bacterial leaf streak in rice, using their major virulence TALEs to physically associate with N-terminal of OsTFIIAγ5. OsTFIIAα and OsTFIIAβ which are post-translationally mature proteins of OsTFIIAαβ separately bound to N- and C-terminal of OsTFIIAγ5. OsTFIIAα coordinated with TALEs for binding with OsTFIIAγ5 to upregulate rice susceptibility genes to cause disease. Conversely, suppression of OsTFIIAαβ attenuated TALEs-targeting genes transcription, thus improved broad-spectrum disease resistance of rice to Xoo and Xoc. These results provide an applicable strategy for improving resistance to TALE-carrying pathogens in rice by appropriate suppression of plant basal transcription factors expression.

Disruption of gene SPL35, encoding a novel CUE domain-containing protein, leads to cell death and enhanced disease response in rice.

SummaryLesion mimic mutants that exhibit spontaneous hypersensitive response (HR)‐like necrotic lesions are ideal experimental systems for elucidating molecular mechanisms involved in plant cell death and defence responses. Here we report identification of a rice lesion mimic mutant, spotted leaf 35 (spl35), and cloning of the causal gene by TAIL‐PCR strategy. spl35 exhibited decreased chlorophyll content, higher accumulation of H2O2, up‐regulated expression of defence‐related marker genes, and enhanced resistance to both fungal and bacterial pathogens of rice. The SPL35 gene encodes a novel CUE (coupling of ubiquitin conjugation to ER degradation) domain‐containing protein that is predominantly localized in cytosol, ER and unknown punctate compartment(s). SPL35 is constitutively expressed in all organs, and both overexpression and knockdown of SPL35 cause the lesion mimic phenotype. SPL35 directly interacts with the E2 protein OsUBC5a and the coatomer subunit delta proteins Delta‐COP1 and Delta‐COP2 through the CUE domain, and down‐regulation of these interacting proteins also cause development of HR‐like lesions resembling those in spl35 and activation of defence responses, indicating that SPL35 may be involved in the ubiquitination and vesicular trafficking pathways. Our findings provide insight into a role of SPL35 in regulating cell death and defence response in plants.

Introduction of the harpinXooc-encoding gene hrf2 in soybean enhances resistance against the oomycete pathogen Phytophthora sojae.

Phytophthora root and stem rot (PRR) caused by an oomycete pathogen Phytophthora sojae is one of the most devastating and widespread diseases throughout soybean-producing regions worldwide. The diversity and variability of P. sojae races make effective control of the pathogen challenging. Here, we introduced an elicitor of plant defense response, the harpinXooc-encoding hrf2 gene from the rice bacterial pathogen Xanthomonas oryzae pv. oryzicola into soybean and evaluated resistance to P. sojae infection. Molecular analysis confirmed the integration and expression of hrf2 in the transgenic soybean. After inoculation with P. sojae, non-transformed control (NC) plants exhibited typical PRR symptoms, including necrotic and wilting leaves, and plant death, whereas most of the transgenic plants showed slightly chlorotic leaves and developed normally. Through T3 to T5 generations, the transgenic events displayed milder disease symptoms and had higher survival rates compared to NC plants, indicating enhanced and stable resistance to P. sojae infection, whereas without P. sojae inoculation, no significant differences in agronomic traits were observed between the transgenic and non-transformed plants. Moreover, after inoculation with P. sojae, significant upregulation of a set of plant defense-related genes, including salicylic acid- and jasmonic acid-dependent and hypersensitive response-related genes was observed in the transgenic plants. Our results indicate that hrf2 expression in transgenic soybean significantly enhanced resistance to P. sojae by eliciting multiple defense responses mediated by different signaling pathways. The potential functional role of the hrf2 gene in plant defense against P. sojae and other pathogens makes it a promising tool for broadening disease resistance in soybean.

Effect of a peroxyacetic acid mixture as green chemical on rice bacterial and fungal pathogens

A peroxyacetic acid mixture (CH3CO3H) has been used as a disinfectant for a low environmental impact. In this study, we investigated whether a commercial peroxyacetic acid mixture, Perosan, can be applied as a green chemical to control bacterial and fungal pathogens, Xanthomonas oryzae pv. oryzae (Xoo) and Rhizoctonia solani in rice plants. Paper discs carrying 25% Perosan caused obvious chlorosis on rice leaves. Ion leakage caused by the phytotoxicity was obviously observed at 5% and higher concentrations of Perosan. Perosan treatment resulted in a slightly decreased pH in rice growing water. In a bacterial growth inhibition assay using optical density measurements, 0.007% of Perosan almost completely inhibited the Xoo growth for 36 h. In an agar dilution assay for antifungal activity, mycelial growth inhibition of R. solani was observed with 0.05% addition of Perosan. In planta antimicrobial activity assay with 5% of Perosan inhibited the lesion lengths in Xoo-inoculated rice plants. A spray of 0.5% Perosan significantly inhibited the lesion developments on the leaves inoculated with an agar disc of R. solani. Our results suggest that Perosan can be applied to rice plants to control the pathogens, Xoo and R. solani, as a green chemical.

Variations in the Structure and Evolution of Rice WRKY Genes in Indica and Japonica Genotypes and their Co-expression Network in Mediating Disease Resistance.

WRKY transcription factor (TF) family regulates many functions in plant growth and development and also during biotic and abiotic stress. In this study, 101 WRKY TF gene models in indica and japonica rice were used to conduct evolutionary analysis, gene structure analysis, and motif composition. Co-expression analysis was carried out first by selecting the differentially expressing genes that showed a significant change in response to the pathogens from Rice Oligonucleotide Array Database (ROAD). About 82 genes showed responses to infection by Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae. Co-expression gene network was constructed using direct neighborhood and context associated inbuilt mode in RiceNetv2 tool. Only 41 genes showed interaction with 2299 non-WRKY genes. Variations exist in the structure and evolution of WRKY genes among indica and japonica genotypes which have important implications in their differential roles including disease resistance. WRKY genes mediate a complex networking and co-express along with other WRKY and non-WRKY genes to mediate resistance against fungal and bacterial pathogens in rice.

Mutagenesis of PhaR, a Regulator Gene of Polyhydroxyalkanoate Biosynthesis of Xanthomonas oryzae pv. oryzae Caused Pleiotropic Phenotype Changes.

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.