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The rice-blast fungus Pyricularia oryzae poses a significant threat to rice production worldwide. Ferroptosis, an iron-dependent form of regulated cell death, has recently been reported to be involved in P. oryzae pathogenicity during plant-fungal interactions. Ferroptosis regulates the developmental cell death of conidia necessary for appressorium maturation. In this study, we have established that a series of benzamides containing a chelating catechol moiety suppresses the formation/maturation of appressoria, which are essential for host infection by the rice blast fungus. Moreover, for the most active compounds we have shown that their activity can be at least partially reversed by adding exogenous Fe 3+ . These results highlight the close association between iron availability and appressorium maturation, opening new avenues for the development of targeted strategies for P. oryzae management.

Dynamic transposition of transposable elements (TEs) in fungal pathogens has significant impact on genome stability, gene expression, and virulence to the host. In Magnaporthe oryzae , genome plasticity resulting from TE insertion is a major driving force leading to the rapid evolution and diversification of this fungus. Despite their importance in M. oryzae population evolution and divergence, our understanding of TEs in this context remains limited. Here, we conducted a genome-wide analysis of TE transposition dynamics in the 11 most abundant TE families in M. oryzae populations. Our results show that these TEs have specifically expanded in recently isolated M. oryzae rice populations, with the presence/absence polymorphism of TE insertions highly concordant with population divergence on Geng/ Japonica and Xian/ Indica rice cultivars. Notably, the genes targeted by clade-specific TEs showed clade-specific expression patterns and are involved in the pathogenic process, suggesting a transcriptional regulation of TEs on targeted genes. Our study provides a comprehensive analysis of TEs in M. oryzae populations and demonstrates a crucial role of recent TE bursts in adaptive evolution and diversification of the M. oryzae rice-infecting lineage., Importance: Magnaporthe oryzae is the causal agent of the destructive blast disease, which caused massive loss of yield annually worldwide. The fungus diverged into distinct clades during adaptation toward the two rice subspecies, Xian/ Indica and Geng/ Japonica . Although the role of TEs in the adaptive evolution was well established, mechanisms underlying how TEs promote the population divergence of M. oryzae remain largely unknown. In this study, we reported that TEs shape the population divergence of M. oryzae by differentially regulating gene expression between Xian/ Indica -infecting and Geng/ Japonica -infecting populations. Our results revealed a TE insertion-mediated gene expression adaption that led to the divergence of M. oryzae population infecting different rice subspecies., Competing Interests: The authors declare no conflict of interest.

Rice blast, caused by Magnaporthe oryzae , is the most destructive rice disease worldwide. The disease symptoms are usually expressed on the leaf and panicle. The leaf disease intensity in controlled environmental conditions is frequently quantified using a 0 to 5 scale, where 0 represents the absence of symptoms, and 5 represents large eyespot lesions. However, this scale restricts the qualitative classification of the varieties into intermediate resistant and susceptible categories. Here, we develop a 0 to 6 scale for blast disease that allows proper assignment of rice breeding lines and varieties into six resistance levels (highly resistant, resistant, moderately resistant, moderately susceptible, susceptible, and highly susceptible). We evaluated 40 common rice varieties against four major blast races (IB1, IB17, IB49, and IE1-K). Varieties carrying the Pi-ta gene were either highly resistant, resistant, or moderately resistant to IB17. The IE1-K race was able to break Pi-ta -mediated resistance of the rice varieties. The Pi-z gene conferred resistance to the IB17 and IE1-K races. The varieties M201, Cheniere, and Frontier were highly susceptible (score 6; 100% disease) to the race IE1-K. Moreover, varieties that were resistant or susceptible to all four blast races also showed similar levels of resistance/susceptibility to blast disease in the field. Taken together, our data proved that the 0 to 6 blast scale can efficiently determine the resistance levels of rice varieties against major blast races. This robust method will assist rice breeding programs to incorporate durable resistance against major and emerging blast races.[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., Competing Interests: The author(s) declare no conflict of interest.

Auxin is an important phytohormone that regulates diverse biologic processes, including plant growth and immunity. Indole-3-acetic acid (IAA), known as one of the main forms of auxin, is able to activate plant immunity. However, it is unknown whether IAA enhances plant resistance and/or suppresses the growth of the fungal pathogen Magnaporthe oryzae . Here, we found that IAA could induce expression levels of pathogenesis-related genes to enhance disease resistance and could control the development of blast disease through inhibiting M. oryzae infection. Exogenous IAA suppressed mycelial growth and delayed spore germination by inhibiting fungal endogenous IAA biosynthesis and impairing redox homeostasis, respectively. When applied to a field test, two IAA analogues, 1-naphthaleneacetic acid and 2,4-dichlorophenoxy acetic acid, can effectively control rice blast disease. Our study advances the understanding of IAA in controlling rice blast disease through suppressing pathogen growth and enhancing plant resistance., Competing Interests: The author(s) declare no conflict of interest.

Rice blast disease, caused by the fungal pathogen Magnaporthe oryzae , is a major threat to rice production worldwide. This study investigates the role of long non-coding RNAs (lncRNAs) in rice's response to this destructive disease, with a focus on their impacts on disease resistance and yield traits. Three specific lncRNAs coded by M. oryzae infection-responsive lncRNAs ( MOIRAs ), MOIRA1 , MOIRA2 , and MOIRA3 , were identified as key regulators of rice's response to M. oryzae infection. Strikingly, when MOIRA1 and MOIRA2 were overexpressed, they exhibited a dual function: they increased rice's susceptibility to blast fungus, indicating a negative role in disease resistance, while simultaneously enhancing tiller numbers and single-plant yield, with no adverse effects on other yield-related traits. This unexpected improvement in productivity suggests the possibility of overcoming the traditional trade-off between disease resistance and crop yield. These findings provide a novel perspective on crop enhancement, offering a promising solution to global food security challenges by developing rice varieties that effectively balance disease resistance and increased productivity.

The data reported here are associated with the article entitled "Analysis of Post-Translational Modification Dynamics Unveiled Novel Insights into Rice Responses to MSP1" [1]. pathogen-associated molecular pattern (PAMP) -triggered immunity (PTI) serves as the fundamental defense mechanism in plants, providing innate protection against pathogen invasion. The fungus Magnaporthe oryzae ( M. oryzae ) secretes MSP1, a protein recognized as a PAMP that induces PTI responses in rice. However, the comprehensive characterization of MSP1-induced post-translational modifications (PTMs) and their contribution to PTI responses remains elusive thus far. In this manuscript, we report the analysis of the phosphoproteome, ubiquitinome, and acetylproteome to investigate the alterations in MSP1-induced changes in these PTMs in MSP1 overexpressed and wild-type rice, utilizing the QExactive TM Orbitrap High-Resolution Mass Spectrometer [1]. Our data primarily focuses on unraveling the PTMs of MSP1-overexpressing transgenic rice, with the goal of elucidating MSP1-induced signaling cascades and deciphering their regulatory mechanisms., (© 2023 The Author(s).)

Weeds that infest crops are a primary factor limiting agricultural productivity worldwide. Weedy rice, also called red rice, has experienced independent evolutionary events through gene flow from wild rice relatives and de-domestication from cultivated rice. Each evolutionary event supplied/equipped weedy rice with competitive abilities that allowed it to thrive with cultivated rice and severely reduce yields in rice fields. Understanding how competitiveness evolves is important not only for noxious agricultural weed management but also for the transfer of weedy rice traits to cultivated rice. Molecular studies of weedy rice using simple sequence repeat (SSR), restriction fragment length polymorphism (RFLP), and whole-genome sequence have shown great genetic variations in weedy rice populations globally. These variations are evident both at the whole-genome and at the single-allele level, including Sh4 (shattering), Hd1 (heading and flowering), and Rc (pericarp pigmentation). The goal of this review is to describe the genetic diversity of current weedy rice germplasm and the significance of weedy rice germplasm as a novel source of disease resistance. Understanding these variations, especially at an allelic level, is also crucial as individual loci that control important traits can be of great target to rice breeders.

Fungal effectors play a pivotal role in suppressing the host defence system, and their evolution is highly dynamic. By comparative sequence analysis of plant-pathogenic fungi and Magnaporthe oryzae, we identified the small secreted C 2 H 2 zinc finger protein MoHTR3. MoHTR3 exhibited high conservation in M. oryzae strains but low conservation among other plant-pathogenic fungi, suggesting an emerging evolutionary selection process. MoHTR3 is exclusively expressed in the biotrophic stage of fungal invasion, and the encoded protein localizes to the biotrophic interfacial complex (BIC) and the host cell nucleus. The signal peptide crucial for MoHTR3' secretion to the BIC and the protein section required for its translocation to the nucleus were both identified by a functional protein domain study. The host-nuclear localization of MoHTR3 suggests a function as a transcriptional modulator of host defence gene induction. After ΔMohtr3 infection, the expression of jasmonic acid- and ethylene-associated genes was diminished in rice, in contrast to when the MoHTR3-overexpressing strain (MoHTR3ox) was applied. The transcript levels of salicylic acid- and defence-related genes were also affected after ΔMohtr3 and MoHTR3ox application. In pathogenicity assays, ΔMohtr3 was indistinguishable from the wild type. However, MoHTR3ox-infected plants showed diminished lesion formation and hydrogen peroxide accumulation, accompanied by a decrease in susceptibility, suggesting that the MoHTR3-induced manipulation of host cells affects host-pathogen interaction. MoHTR3 emphasizes the role of the host nucleus as a critical target for the pathogen-driven manipulation of host defence mechanisms and underscores the ongoing evolution of rice blast's arms race., (© 2023 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

A subset of plant intracellular NLR immune receptors detect effector proteins, secreted by phytopathogens to promote infection, through unconventional integrated domains which resemble the effector's host targets. Direct binding of effectors to these integrated domains activates plant defenses. The rice NLR receptor Pik-1 binds the Magnaporthe oryzae effector AVR-Pik through an integrated heavy metal-associated (HMA) domain. However, the stealthy alleles AVR-PikC and AVR-PikF avoid interaction with Pik-HMA and evade host defenses. Here, we exploited knowledge of the biochemical interactions between AVR-Pik and its host target, OsHIPP19, to engineer novel Pik-1 variants that respond to AVR-PikC/F. First, we exchanged the HMA domain of Pikp-1 for OsHIPP19-HMA, demonstrating that effector targets can be incorporated into NLR receptors to provide novel recognition profiles. Second, we used the structure of OsHIPP19-HMA to guide the mutagenesis of Pikp-HMA to expand its recognition profile. We demonstrate that the extended recognition profiles of engineered Pikp-1 variants correlate with effector binding in planta and in vitro, and with the gain of new contacts across the effector/HMA interface. Crucially, transgenic rice producing the engineered Pikp-1 variants was resistant to blast fungus isolates carrying AVR-PikC or AVR-PikF. These results demonstrate that effector target-guided engineering of NLR receptors can provide new-to-nature disease resistance in crops., Competing Interests: JM, MS, AB, SV, MF, AL, CS, JD, AB, RT, MB No competing interests declared, SK receives funding from industry on NLR biology, (© 2023, Maidment et al.)

Agrobacterium -mediated transient expression (AMTE) has been widely used for high-throughput assays of gene function in diverse plant species. However, its application in monocots is still limited due to low expression efficiency. Here, by using histochemical staining and a quantitative fluorescence assay of β-glucuronidase (GUS) gene expression, we investigated factors affecting the efficiency of AMTE on intact barley plants. We found prominent variation in GUS expression levels across diverse vectors commonly used for stable transformation and that the vector pCBEP produced the highest expression. Additionally, concurrent treatments of plants with one day of high humidity and two days of darkness following agro-infiltration also significantly increased GUS expression efficiency. We thus established an optimized method for efficient AMTE on barley and further demonstrated its efficiency on wheat and rice plants. We showed that this approach could produce enough proteins suitable for split-luciferase assays of protein-protein interactions on barley leaves. Moreover, we incorporated the AMTE protocol into the functional dissection of a complex biological process such as plant disease. Based on our previous research, we used the pCBEP vector to construct a full-length cDNA library of genes upregulated during the early stage of rice blast disease. A subsequent screen of the library by AMTE identified 15 candidate genes (out of ~2000 clones) promoting blast disease on barley plants. Four identified genes encode chloroplast-related proteins: OsNYC3, OsNUDX21, OsMRS2-9, and OsAk2. These genes were induced during rice blast disease; however, constitutive overexpression of these genes conferred enhanced disease susceptibility to Colletotrichum higginsianum in Arabidopsis . These observations highlight the power of the optimized AMTE approach on monocots as an effective tool for facilitating functional assays of genes mediating complex processes such as plant-microbe interactions.

During effector-triggered immunity (ETI) against the devastating rice blast pathogen Magnaporthe oryzae, Pi9 functions as an intracellular resistance protein sensing the pathogen-secreted effector AvrPi9 in rice. Importantly, the underlying recognition mechanism(s) between Pi9 and AvrPi9 remains elusive. In this study, we identified a rice ubiquitin-like domain-containing protein (UDP), AVRPI9-INTERACTING PROTEIN 1 (ANIP1), which is directly targeted by AvrPi9 and also binds to Pi9 in plants. Phenotypic analysis of anip1 mutants and plants overexpressing ANIP1 revealed that ANIP1 negatively modulates rice basal defense against M. oryzae. ANIP1 undergoes 26S proteasome-mediated degradation, which can be blocked by both AvrPi9 and Pi9. Moreover, ANIP1 physically associates with the rice WRKY transcription factor OsWRKY62, which also interacts with AvrPi9 and Pi9 in plants. In the absence of Pi9, ANIP1 negatively regulates OsWRKY62 abundance, which can be promoted by AvrPi9. Accordingly, knocking out of OsWRKY62 in a non-Pi9 background decreased immunity against M. oryzae. However, we also observed that OsWRKY62 plays negative roles in defense against a compatible M. oryzae strain in Pi9-harboring rice. Pi9 binds to ANIP1 and OsWRKY62 to form a complex, which may help to keep Pi9 in an inactive state and weaken rice immunity. Furthermore, using competitive binding assays, we showed that AvrPi9 promotes Pi9 dissociation from ANIP1, which could be an important step toward ETI activation. Taken together, our results reveal an immune strategy whereby a UDP-WRKY module, targeted by a fungal effector, modulates rice immunity in distinct ways in the presence or absence of the corresponding resistance protein., (Copyright © 2023 The Author. Published by Elsevier Inc. All rights reserved.)

Functional characterization of stress-responsive genes through the analysis of transgenic plants is a standard approach to comprehend their role in climate resilience and subsequently exploit them for sustainable crop improvement. In this study, we investigated the function of LOC_Os04g59420 , a gene of DUF740 family ( OsSRDP - Oryza sativa Stress Responsive DUF740 Protein) from rice, which showed upregulation in response to abiotic stress in the available global expression data, but is yet to be functionally characterized. Transgenic plants of the rice OsSRDP gene, driven by a stress-inducible promoter AtRd29A , were developed in the background of cv. Pusa Sugandh 2 (PS2) and their transgene integration and copy number were confirmed by molecular analysis. The three independent homozygous transgenic plants (AtRd29A::OsSRDP rice transformants) showed better resilience to drought, salinity, and cold stresses, but not heat stress, as compared to the non-transformed PS2, which corresponded with their respective relative transcript abundance for OsSRDP . Transgenic plants maintained higher RWC, photosynthetic pigments, and proline accumulation under drought and salinity stresses. Furthermore, they exhibited less accumulation of reactive oxygen species (ROS) than PS2 under drought stress, as seen from the transcript abundance studies of the ROS genes. Under cold stress, OsSRDP transgenic lines illustrated minimal cell membrane injury compared to PS2. Additionally, the transgenic plants showed resistance to a virulent strain of rice blast fungus, Magnaporthe oryzae ( M. oryzae ). The promoter analysis of the gene in N22 and PS2 revealed the presence of multiple abiotic and biotic stress-specific motif elements supporting our observation on multiple stress tolerance. Based on bioinformatics studies, we identified four potential candidate interaction partners for LOC_Os04g59420 , of which two genes ( LOC_Os05g09640 and LOC_Os06g50370 ) showed co-expression under biotic and drought stress along with OsSRDP . Altogether, our findings established that stress-inducible expression of OsSRDP can significantly enhance tolerance to multiple abiotic stresses and a biotic stress., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jayaraman, Sevanthi, Raman, Jiwani, Solanke, Mandal and Mohapatra.)

Data reported here is associated with the article entitled "TMT-based quantitative membrane proteomics identified pattern recognition receptors (PRRs) potentially involved in the perception of MSP1 in rice leaves" [1]. PAMP-triggered immunity (PTI) constitutes the first layer of plant innate immunity against pathogen infection. M. oryzae secreted protein MSP1 has been identified as a PAMP which induces PTI responses in rice. However, identification of PRRs involved in the recognition of MSP1 has not been achieved so far. In this manuscript, we carried out comprehensive proteomic profiling to investigate the potential PRRs and MSP1 induced signaling cascades using MSP1 overexpressed transgenic rice by TMT-labeling based quantitative analysis with QExactive TM Orbitrap High-Resolution Mass Spectrometer [1]., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Significance: Circadian-controlled cellular growth, differentiation, and metabolism are mainly achieved by a classical transcriptional-translational feedback loop (TTFL), as revealed by investigations in animals, plants, and fungi. Recent Advances: Recently, reactive oxygen species (ROS) have been reported as part of a cellular network synchronizing nontranscriptional oscillators with established TTFL components, adding complexity to regulatory mechanisms of circadian rhythm. Both circadian rhythm and ROS homeostasis have a great impact on plant immunity as well as fungal pathogenicity, therefore interconnections of these two factors are implicit in plant-fungus interactions. Critical Issues: In this review, we aim to summarize the recent advances in circadian-controlled ROS homeostasis, or ROS-modulated circadian clock, in plant-fungus pathosystems, particularly using the rice ( Oryza sativa ) blast fungus ( Magnaporthe oryzae ) pathosystem as an example. Understanding of such bidirectional interaction between the circadian timekeeping machinery and ROS homeostasis/signaling would provide a theoretical basis for developing disease control strategies for important plants/crops. Future Directions: Questions remain unanswered about the detailed mechanisms underlying circadian regulation of redox homeostasis in M. oryzae , and the consequent fungal differentiation and death in a time-of-day manner. We believe that the rice- M. oryzae pathobiosystem would provide an excellent platform for investigating such issues in circadian-ROS interconnections in a plant-fungus interaction context. Antioxid. Redox Signal . 37, 726-738.

Endo-β-1,4-Xylanases are a group of extracellular enzymes that catalyze the hydrolysis of xylan, a principal constituent of the plant primary cell wall. The contribution of Endo-β-1,4-Xylanase I to both physiology and pathogenesis of the rice blast fungus M. oryzae is unknown. Here, we characterized the biological function of two endoxylanase I (MoXYL1A and MoXYL1B) genes in the development of M. oryzae using targeted gene deletion, biochemical analysis, and fluorescence microscopy. Phenotypic analysis of ∆Moxyl1A strains showed that MoXYL1A is required for the full virulence of M. oryzae but is dispensable for the vegetative growth of the rice blast fungus. MoXYL1B, in contrast, did not have a clear role in the infectious cycle but has a critical function in asexual reproduction of the fungus. The double deletion mutant was severely impaired in pathogenicity and virulence as well as asexual development. We found that MoXYL1A deletion compromised appressorium morphogenesis and function, leading to failure to penetrate host cells. Fluorescently tagged MoXYL1A and MoXYL1B displayed cytoplasmic localization in M. oryzae, while analysis of MoXYL1A-GFP and MoXYL1B-GFP in-planta revealed translocation and accumulation of these effector proteins into host cells. Meanwhile, sequence feature analysis showed that MoXYL1A possesses a transient chloroplast targeting signal peptide, and results from an Agrobacterium infiltration assay confirmed co-localization of MoXYL1A-GFP with ChCPN10C-RFP in the chloroplasts of host cells. MoXYL1B, accumulated to the cytoplasm of the host. Taken together, we conclude that MoXYL1A is a secreted effector protein that likely promotes the virulence of M. oryzae by interfering in the proper functioning of the host chloroplast, while the related xylanase MoXYL1B does not have a major role in virulence of M. oryzae., (© 2022. The Author(s).)

Successful cultivation of rice (Oryza sativa L.) in many Asian countries requires submergence stress tolerance at the germination and early establishment stages. Two quantitative trait loci, Sub1 (conferring submergence tolerance) and AG1 (conferring anaerobic germination), were recently pyramided into a single genetic background, without compromising any desirable agronomic traits, leading to the development of Ciherang-Sub1 + AG1 (CSA). However, little research has been conducted to enhance plant tolerance to abiotic stress (submergence) and biotic stress (rice blast), which occur in a damp climate following flooding. The BC 2 F 5 breeding line was phenotypically characterized using the AvrPi9 isolate. The biotic and abiotic stress tolerance of selected lines was tested under submergence stress and anaerobic germination conditions, and lines tolerant to each stress condition were identified through phenotypic and gene expression analyses. The Ciherang-Sub1 + AG1 + Pi9 (CSA-Pi9) line showed similar agronomic performance to its recurrent parent, CSA, but had significantly reduced chalkiness in field trials conducted in temperate regions. Unexpectedly, the CSA-Pi9 line also showed salinity tolerance. Thus, the breeding line newly developed in this study, CSA-Pi9, functioned under stress conditions, in which Sub1, AG1, and Pi9 play a role and had superior grain quality traits compared to its recurrent parent in temperate regions. We speculate that CSA-Pi9 will enable the establishment of climate-resilient rice cropping systems, particularly in East Asia., (© 2022. The Author(s).)

Oxysterol-binding protein-related proteins (ORPs) are a conserved class of lipid transfer proteins that are closely involved in multiple cellular processes in eukaryotes, but their roles in plant-pathogen interactions are mostly unknown. We show that transient expression of ORPs of Magnaporthe oryzae (MoORPs) in Nicotiana benthamina plants triggered oxidative bursts and cell death; treatment of tobacco Bright Yellow-2 suspension cells with recombinant MoORPs elicited the production of reactive oxygen species. Despite ORPs being normally described as intracellular proteins, we detected MoORPs in fungal culture filtrates and intercellular fluids from barley plants infected with the fungus. More importantly, infiltration of Arabidopsis plants with recombinant Arabidopsis or fungal ORPs activated oxidative bursts, callose deposition, and PR1 gene expression, and enhanced plant disease resistance, implying that ORPs may function as endogenous and exogenous danger signals triggering plant innate immunity. Extracellular application of fungal ORPs exerted an opposite impact on salicylic acid and jasmonic acid/ethylene signaling pathways. Brassinosteroid Insensitive 1-associated Kinase 1 was dispensable for the ORP-activated defense. Besides, simultaneous knockout of MoORP1 and MoORP3 abolished fungal colony radial growth and conidiation, whereas double knockout of MoORP1 and MoORP2 compromised fungal virulence on barley and rice plants. These observations collectively highlight the multifaceted role of MoORPs in the modulation of plant innate immunity and promotion of fungal development and virulence in M. oryzae., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Rice blast disease is one of the major bottlenecks of rice production in the world including Bangladesh. To develop blast resistant lines, a cross was made between a high yielding but blast susceptible variety MR263 and a blast resistant variety Pongsu seribu 2. Marker-assisted backcross breeding was followed to develop F 1 , BC 1 F 1 , BC 2 F 1 , BC 2 F 2 , BC 2 F 3, BC 2 F 4 and BC 2 F 5 population. DNA markers i.e., RM206, RM1359 and RM8225 closely linked to Pb1, pi21 and Piz blast resistant genes, respectively and marker RM276 linked to panicle blast resistant QTL ( qPbj-6.1 ) were used in foreground selection . Calculated chi-square (χ 2 ) value of phenotypic and genotypic segregation data of BC 2 F 1 population followed goodness of fit to the expected ratio (1:1) (phenotypic data χ 2  = 1.08, p  = 0.701; genotypic data χ 2  = range from 0.33 to 3.00, p  = 0.08-0.56) and it indicates that the inheritance pattern of blast resistance was followed by a single gene model. Eighty-nine advanced lines of BC 2 F 5 population were developed and out of them, 58 lines contained Piz, Pb1, pi21, and qPbj-6.1 while 31 lines contained Piz, Pb1, and QTL qPbj-6.1 . Marker-trait association analysis revealed that molecular markers i.e., RM206, RM276, and RM8225 were tightly linked with blast resistance, and each marker was explained by 33.33% phenotypic variation (resistance reaction). Morphological and pathogenicity performance of advanced lines was better compared to the recurrent parent. Developed blast resistance advanced lines could be used as donors or blast resistant variety for the management of devastating rice blast disease., Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01141-3., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© Prof. H.S. Srivastava Foundation for Science and Society 2022.)

Acetylation and deacetylation of histones are key epigenetic mechanisms for gene regulation in response to environmental stimuli. RPD3 is a well-conserved class I histone deacetylase (HDAC) that is involved in diverse biological processes. Here, we investigated the roles of the Magnaporthe oryzae RPD3 ( MoRPD3 ) gene, an ortholog of Saccharomyces cerevisiae Rpd3 , during development and pathogenesis in the model plant-pathogenic fungus Magnaporthe oryzae. We demonstrated that the MoRPD3 gene is able to functionally complement the yeast Rpd 3 deletion mutant despite the C-terminal extension of the MoRPD3 protein. MoRPD3 localizes primarily to the nuclei of vegetative hyphae, asexual spores, and invasive hyphae. Deletion of MoRPD3 appears to be lethal. Depletion of MoRPD3 transcripts via gene silencing ( MoRPD3 kd , where " kd " stands for "knockdown") has opposing effects on asexual and sexual reproduction. Although conidial germination and appressorium formation rates of the mutants were almost comparable to those of the wild type, in-depth analysis revealed that the appressoria of mutants are smaller than those of the wild type. Furthermore, the MoRPD3 kd strain shows a significant reduction in pathogenicity, which can be attributed to the delay in appressorium-mediated penetration and impaired invasive growth. Interestingly, MoRPD3 does not regulate potassium transporters, as shown for Rpd3 of S. cerevisiae. However, it functioned in association with the target of rapamycin (TOR) kinase pathway, resulting in the dependency of appressorium formation on hydrophilic surfaces and on TOR's inhibition by MoRPD3. Taken together, our results uncovered distinct and evolutionarily conserved roles of MoRPD3 in regulating fungal reproduction, infection-specific development, and virulence. IMPORTANCE RPD3 is an evolutionarily conserved class I histone deacetylase (HDAC) that plays a pivotal role in diverse cellular processes. In filamentous fungal pathogens, abrogation of the gene encoding RPD3 results in either lethality or severe growth impairment, making subsequent genetic analyses challenging. Magnaporthe oryzae is a causal agent of rice blast disease, which is responsible for significant annual yield losses in rice production. Here, we characterized the RPD3 gene of M. oryzae ( MoRPD3 ) in unprecedented detail using a gene-silencing approach. We provide evidence that MoRPD3 is a bona fide HDAC regulating fungal reproduction and pathogenic development by potentially being involved in the TOR-mediated signaling pathway. To the best of our knowledge, this work is the most comprehensive genetic dissection of RPD3 in filamentous fungal pathogens. Our work extends and deepens our understanding of how an epigenetic factor is implicated in the development and virulence of fungal pathogens of plants.

Rice blast disease caused by the filamentous Ascomycetous fungus Magnaporthe oryzae is a major threat to rice production worldwide. The mechanisms underlying rice resistance to M. oryzae, such as transcriptional reprogramming and signalling networks, remain elusive. In this study, we carried out an in-depth comparative transcriptome study on the susceptible and resistant rice cultivars in response to M. oryzae. Our analysis highlighted that rapid, high-amplitude transcriptional reprogramming was important for rice defence against blast fungus. Ribosome- and protein translation-related genes were significantly enriched among differentially expressed genes (DEGs) at 12 hpi in both cultivars, indicating that the protein translation machinery is regulated in the activation of immunity in rice. Furthermore, we identified a core set of genes that are involved in the rice response to both biotic and abiotic stress. More importantly, among the core genes, we demonstrated that the metallothionein OsMT1a and OsMT1b genes positively regulated rice resistance while a peroxidase gene Perox4 negatively regulated rice resistance to M. oryzae. Our study provides novel insight into transcriptional reprogramming and serves as a valuable resource for functional studies on rice immune signalling components in resistance to blast disease., (© 2021 John Wiley & Sons Ltd.)

Blast is one of the most devastating fungal diseases of rice caused by Magnaporthe oryzae. Plant essential oil (EO) can function as antifungal agents and are regarded as a safe and acceptable method for plant disease control. However, EOs are unstable and hydrophobic, which limits its use. In the present study, we aimed for the preparation and characterization of a nanoemulsion (NE) from green tea essential oil (GTO) by ultrasonication method and determined the antifungal activity of NE onM. oryzae. The particle size and zeta potential of the NE were 86.98 nm and -15.1 mV, respectively. The chemical composition and functional groups of GTO and NE were studied by using GC-MS analysis, portable Raman spectroscopy, and FTIR coupled with chemometric analysis. GC-MS analysis showed the major components in GTO and NE were n-Hexyl cinnamaldehyde and L-α-Terpineol. Both GTO and NE showed good antioxidant activity and total phenol content. Moreover, the NE showed good antifungal activity againstM. oryzae which was further confirmed by scanning electron microscopy (SEM) examination. Also, confocal Raman micro-spectroscopy (CRM) revealed the antifungal mechanism of GTO and NE on M. oryzae which proves the cell damage. To the best of our knowledge, this is the first study on the antifungal activity of GTO and NE against M. oryzae and also the use of CRM for the evaluation of the chemical changes in single fungal hyphae in a holistic approach. This study suggests that the prepared NE could be a potential candidate for use as a substitute for synthetic fungicides., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Rice blast disease is one of the most common rice diseases worldwide. It is essential to improve disease resistance through environment-friendly methods, while maintaining yield and quality parameters. In this study, jasmonic acid (JA), a plant hormone with anti-fungal activity, was obtained, at both low (100 μmol/L) and high (400 μmol/L) concentrations in rice leaves, before, during, and after infection, respectively. JA could inhibit germination and appressorium formation of rice blast spores in a dose-dependent manner. A total of 400-μmol/L JA treatment significantly enhanced cell viability and endogenous JA level in rice leaves. Furthermore, rice leaves inoculated with Magnaporthe oryzae and sprayed with JA 72 h post-inoculation showed the maximum symptom relief and the highest endogenous JA production among all treatment approaches. The expressions of defense-related genes, OsPR10a and OsAOS2, were highly up-regulated in response to JA, whereas OsEDS1 was down-regulated. Hence, we revealed that exogenous JA could activate JA signaling to effectively control the symptoms of rice blast.

Background: Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases of rice. An increasing number of microRNAs (miRNAs) have been reported to fine-tune rice immunity against M. oryzae and coordinate with growth and development., Results: Here, we showed that rice microRNA159a (Osa-miR159a) played a positive role in rice resistance to M. oryzae. The expression of Osa-miR159a was suppressed in a susceptible accession at 12, 24, and 48 h post-inoculation (hpi); it was upregulated in a resistant accession of M. oryzae at 24 hpi. The transgenic rice lines overexpressing Osa-miR159a were highly resistant to M. oryzae. In contrast, the transgenic lines expressing a short tandem target mimic (STTM) to block Osa-miR159a showed enhanced susceptibility. Knockout mutations of the target genes of Osa-miR159a, including OsGAMYB, OsGAMYBL, and OsZF, led to resistance to M. oryzae. Alteration of the expression of Osa-miR159a impacted yield traits including pollen and grain development., Conclusions: Our results indicated that Osa-miR159a positively regulated rice immunity against M. oryzae by downregulating its target genes. Proper expression of Osa-miR159a was critical for coordinating rice blast resistance with grain development.

Rice cultivars from japonica and indica lineage possess differential resistance against blast fungus as a result of genetic divergence. Whether different rice cultivars also show distinct metabolomic changes in response to P. oryzae, and their role in host resistance, are poorly understood. Here, we examine the responses of six different rice cultivars from japonica and indica lineage challenged with P. oryzae. Both susceptible and resistant rice cultivars expressed several metabolites exclusively during P. oryzae infection, including the saponin Bayogenin 3-O-cellobioside. Bayogenin 3-O-cellobioside level in infected rice directly correlated with their resistant attributes. These findings reveal, for the first time to our knowledge that besides oat, other grass plants including rice produces protective saponins. Our study provides insight into the role of pathogen-mediated metabolomics reprogramming in host immunity. The correlation between Bayogenin 3-O-Cellobioside levels and blast resistance suggests that engineering saponin expression in cereal crops represents attractive and sustainable disease management., (© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Synthesis of assumed natural (12R,13S)-enantiomers of pyriculariol (1) and dihydropyriculariol (2), phytotoxins isolated from rice blast disease fungus, Pyricularia oryzae, was achieved using Wittig reaction or microwave-assisted Stille coupling reaction as the key step. The synthesis revealed that the natural 1 and 2 are racemates. Foliar application test on a rice leaf indicated that both the salicylaldehyde core and side chain were necessary for phytotoxic activity. The fungus is found to produce optically active phytotoxins when incubated with rotary shaker, but racemic ones when cultured using an aerated jar fermenter., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Background: Long non-coding RNAs (LncRNAs) have emerged as important regulators in many physiological processes in plant. By high-throughput RNA-sequencing, many pathogen-associated LncRNAs were mapped in various plants, and some of them were proved to be involved in plant defense responses. The rice blast disease caused by Magnaporthe oryzae (M. oryzae) is one of the most destructive diseases in rice. However, M. oryzae-induced LncRNAs in rice is yet to be studied., Findings: We investigated rice LncRNAs that were associated with the rice blast fungus. Totally 83 LncRNAs were up-regulated after blast fungus infection and 78 were down-regulated. Of them, the natural antisense transcripts (NATs) were the most abundant. The expression of some LncRNAs has similar pattern with their host genes or neighboring genes, suggesting a cis function of them in regulating gene transcription level. The deferentially expressed (DE) LncRNAs and genes co-expression analysis revealed some LncRNAs were associated with genes known to be involved in pathogen resistance, and these genes were enriched in terpenoid biosynthesis and defense response by Gene Ontology (GO) enrichment analysis. Interestingly, one of up-regulated DE-intronic RNA was derived from a jasmonate (JA) biosynthetic gene, lipoxygenase RLL (LOX-RLL). Levels of JAs were significantly increased after blast fungus infection. Given that JA is known to regulate blast resistance in rice, we suggested that LncRNA may be involved in JA-mediated rice resistance to blast fungus., Conclusions: This study identified blast fungus-responsive LncRNAs in rice, which provides another layer of candidates that regulate rice and blast fungus interactions.

Availability and efficient utilization of host-derived nutrients by pathogens decide the fate of host-pathogen interaction. In Magnaporthe oryzae, N-acetylglucosamine (GlcNAc) catabolic pathway was found essential for successful host colonization and pathogenicity. GlcNAc catabolic enzymes hexokinase, GlcNAc-6-phosphate deacetylase (MoDac) and GlcN-6-phosphate deaminase (MoDeam) are encoded in a genomic cluster in M. oryzae and several phytopathogenic fungi. However, transcriptional regulation of GlcNAc catabolic pathway was not understood. We identified a conserved Ndt80/PhoG-like transcriptional regulator as a part of the GlcNAc catabolic gene cluster in M. oryzae and other fungi. We found that MoNdt80 is essential for GlcNAc utilization and pathogenicity of M. oryzae. Unlike WT, ΔMoNdt80 failed to induce transcription of GlcNAc catabolic pathway genes in response to GlcNAc. MoNdt80 could bind to a specific cis-acting consensus sequence GNCRCAAA[AT], present in the promoter of MoDac, MoDeam and β-hexosaminidase (MoHex). Further, comparative RNA-sequencing analysis using WT and ΔMoNdt80 revealed a large set of GlcNAc responsive genes that are under the transcriptional control of MoNdt80. These genes encoded GlcNAc catabolic enzymes, transporters and cell wall degrading enzymes which are required for hyphal growth expansion during host colonization. Overall, these results suggest MoNdt80 mediated transcriptional regulation of GlcNAc catabolic pathway is essential for successful host colonization and pathogenesis., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

The exocyst, an evolutionarily conserved octameric protein complex, mediates tethering of vesicles to the plasma membrane in the early stage of exocytosis. Arabidopsis Exo70, a subunit of the exocyst complex, has been found to be involved in plant immunity. Here, we characterize the function of OsExo70B1 in rice. OsExo70B1 mainly expresses in leaf and shoot and its expression is induced by pathogen-associated molecular patterns (PAMPs) and rice blast fungus Magnaporthe oryzae ( M. oryzae ). Knocking out OsExo70B1 results in significantly decreased resistance and defense responses to M. oryzae compared to the wild type, including more disease lesions and enhanced fungal growth, downregulated expression of pathogenesis-related (PR) genes, and decreased reactive oxygen species accumulation. In contrast, the exo70B1 mutant does not show any defects in growth and development. Furthermore, OsExo70B1 can interact with the receptor-like kinase OsCERK1, an essential component for chitin reception in rice. Taken together, our data demonstrate that OsExo70B1 functions as an important regulator in rice immunity.

Rice blast caused by Magnaporthe oryzae is one of the most serious rice diseases worldwide. Biological control is gaining popularity as a promising method for the control of this disease; however, more effective microbial strains with strong adaptability in rice fields need to be identified. Here, we report for the first time the successful identification of biocontrol bacterial strains from frozen soils of the soda saline-sodic land. We isolated 82 bacterial strains from rice fields in the western Songnen Plain of China, one of the three major soda saline soils in the world. Five of the isolated strains exhibited strong inhibition to M. oryzae growth. The potential strains were identified as Bacillus safensis JLS5, Pseudomonas koreensis JLS8, Pseudomonas saponiphila JLS10, Stenotrophomonas rhizophila JLS11 and Bacillus tequilensis JLS12, respectively, by 16s RNA gene sequence analysis. The antagonistic assay and the artificial inoculation tests showed that JLS5 and JLS12 could effectively inhibit conidial germination and pathogenicity of the rice blast fungus, both preventively and curatively. The suppression of pathogenicity was further confirmed by greenhouse experiments, showing the effectiveness of JLS5 and JLS12 as a potential biological control agents of M. oryzae . The potential application of these cold-tolerant strains for rice blast control in cold regions is discussed. Our data suggest that soda saline-sodic soils are a rich source for biocontrol strain isolation.

Jao Hom Nin (JHN) is a Thai rice variety with broad-spectrum resistant against rice blast fungus. JHN contains two rice blast resistant genes, Pish and Pik , located on chromosome 1 and on chromosome 11, respectively. To understand the blast resistance in JHN, the study of the defense mechanism related to the Pish and Pik genes is crucial. This study aimed to dissect defense response genes between the Pish and Pik genes using the RNA-seq technique. Differentially expressed genes (DEGs) of Pish and Pik backcross inbred lines were identified between 0 and 24 h after inoculation with rice blast spore suspension. The results showed that 1248 and 858 DEGs were unique to the Pish and Pik lines, respectively. The wall-associated kinase gene was unique to the Pish line and the zinc-finger-containing protein gene was unique to the Pik line. Pathogenicity-related proteins PR-4 and PR-10 were commonly found in both Pish and Pik lines. Moreover, DEGs functionally categorized in brassinosteriod, jasmonic acid, and salicylic acid pathways were detected in both Pish and Pik lines. These unique and shared genes in the Pish and Pik rice blast defense responses will help to dissect the mechanisms of plant defense and facilitate rice blast breeding programs.

Rhizosphere bacteria can positively influence plant growth by direct and indirect mechanisms. A total of 112 bacterial strains were isolated from the rhizosphere of rice and tested for plant beneficial activities such as siderophore production, cell-wall-degrading enzyme production, hydrogen cyanide (HCN) production and antifungal activity against rice blast disease fungus. The actinomycetes count was 3.8 × 10 6 CFU/g soil. Streptomyces strains PC 12, D 4.1, D 4.3 and W1 showed strong growth inhibition of blast disease fungus, Pyricularia sp. (87.3%, 82.2%, 80.0% and 80.5%) in vitro. Greenhouse experiments revealed that rice plants treated with Streptomyces strain PC 12 recorded maximum plant height, root length and root dry weight compared to the control. Taxonomic characterization of this strain on the basis of 16S rRNA gene sequence led to its identification as Streptomyces palmae PC 12. Streptomyces palmae PC 12 may be used as biofertilizer to enhance the growth and productivity of commercially important rice cultivar RD6 and the biocontrol of blast disease fungus., Competing Interests: The authors declare no conflict of interest.

Magnaporthe grisea is the causal agent of rice blast disease, which is the most serious disease of cultivated rice. Aromatic polyketides are its typical metabolites and are involved in the infection process. In the search for novel lead compounds, chemical investigation of the fungus M. grisea M639 has led to the isolation of four new aromatic polyketides (salicylaldehyde skeleton bearing an unsaturated side chain), griseaketides A-D ( 1 - 4 ), as well as 15 known compounds ( 5 - 19 ). The structures of the new compounds were elucidated on the basis of extensive spectroscopic analyses, including HR-MS, 2D NMR. Compound 12 showed prominent activity that killed 94.5% of C. elegans at 400 ppm and 66.9% at 200 ppm over 24 h. This is the first report describing the nematicidal activity of this type aromatic polyketide.

Histone acetylation/deacetylation represent a general and efficient epigenetic mechanism through which fungal cells control gene expression. Here we report developmental requirement of MoHOS2-mediated histone deacetylation (HDAC) for the rice blast fungus, Magnaporthe oryzae. Structural similarity and nuclear localization indicated that MoHOS2 is an ortholog of Saccharomyces cerevisiae Hos2, which is a member of class I histone deacetylases and subunit of Set3 complex. Deletion of MoHOS2 led to 25% reduction in HDAC activity, compared to the wild-type, confirming that it is a bona-fide HDAC. Lack of MoHOS2 caused decrease in radial growth and impinged dramatically on asexual sporulation. Such reduction in HDAC activity and phenotypic defects of ΔMohos2 were recapitulated by a single amino acid change in conserved motif that is known to be important for HDAC activity. Expression analysis revealed up-regulation of MoHOS2 and concomitant down-regulation of some of the key genes involved in asexual reproduction under sporulation-promoting condition. In addition, the deletion mutant exhibited defect in appressorium formation from both germ tube tip and hyphae. As a result, ΔMohos2 was not able to cause disease symptoms. Wound-inoculation showed that the mutant is compromised in its ability to grow inside host plants as well. We found that some of ROS detoxifying genes and known effector genes are de-regulated in the mutant. Taken together, our data suggest that MoHOS2-dependent histone deacetylation is pivotal for proper timing and induction of transcription of the genes that coordinate developmental changes and host infection in M. oryzae.

Rice blast disease, caused by Magnaporthe oryzae , is one of the major constraints to rice production, which feeds half of the world's population. Proteomic technologies have been used as effective tools in plant-pathogen interactions to study the biological pathways involved in pathogen infection, plant response, and disease progression. Advancements in mass spectrometry (MS) and apoplastic and plasma membrane protein isolation methods facilitated the identification and quantification of subcellular proteomes during plant-pathogen interaction. Proteomic studies conducted during rice- M. oryzae interaction have led to the identification of several proteins eminently involved in pathogen perception, signal transduction, and the adjustment of metabolism to prevent plant disease. Some of these proteins include receptor-like kinases (RLKs), mitogen-activated protein kinases (MAPKs), and proteins related to reactive oxygen species (ROS) signaling and scavenging, hormone signaling, photosynthesis, secondary metabolism, protein degradation, and other defense responses. Moreover, post-translational modifications (PTMs), such as phosphoproteomics and ubiquitin proteomics, during rice- M. oryzae interaction are also summarized in this review. In essence, proteomic studies carried out to date delineated the molecular mechanisms underlying rice- M. oryzae interactions and provided candidate proteins for the breeding of rice blast resistant cultivars., (Copyright © 2019 Meng, Gupta, Min, Kwon, Wang, Je, Kim, Jeon, Agrawal, Rakwal and Kim.)

Unconventional integrated domains in plant intracellular immune receptors of the nucleotide-binding leucine-rich repeat (NLRs) type can directly bind translocated effector proteins from pathogens and thereby initiate an immune response. The rice ( Oryza sativa ) immune receptor pairs Pik-1/Pik-2 and RGA5/RGA4 both use integrated heavy metal-associated (HMA) domains to bind the effectors AVR-Pik and AVR-Pia, respectively, from the rice blast fungal pathogen Magnaporthe oryzae These effectors both belong to the MAX effector family and share a core structural fold, despite being divergent in sequence. How integrated domains in NLRs maintain specificity of effector recognition, even of structurally similar effectors, has implications for understanding plant immune receptor evolution and function. Here, using plant cell death and pathogenicity assays and protein-protein interaction analyses, we show that the rice NLR pair Pikp-1/Pikp-2 triggers an immune response leading to partial disease resistance toward the "mis-matched" effector AVR-Pia in planta and that the Pikp-HMA domain binds AVR-Pia in vitro We observed that the HMA domain from another Pik-1 allele, Pikm, cannot bind AVR-Pia, and it does not trigger a plant response. The crystal structure of Pikp-HMA bound to AVR-Pia at 1.9 Å resolution revealed a binding interface different from those formed with AVR-Pik effectors, suggesting plasticity in integrated domain-effector interactions. The results of our work indicate that a single NLR immune receptor can bait multiple pathogen effectors via an integrated domain, insights that may enable engineering plant immune receptors with extended disease resistance profiles., (© 2019 Varden et al.)

Background: Rice (Oryza sativa L.) is one of the most important crops that serves as staple food for ~ 50% of the human population worldwide. Some important agronomic traits that allow rice to cope with numerous abiotic and biotic stresses have been selected and fixed during domestication. Knowledge on how expression divergence of genes gradually contributes to phenotypic differentiation in response to biotic stress and their contribution to rice population speciation is still limited., Results: Here, we explored gene expression divergence between a japonica rice cultivar Nipponbare and an indica rice cultivar 93-11 in response to invasion by the filamentous ascomycete fungus Magnaporthe oryzae (Pyricularia oryzae), a plant pathogen that causes significant loss to rice production worldwide. We investigated differentially expressed genes in the two cultivars and observed that evolutionarily conserved orthologous genes showed highly variable expression patterns under rice blast infection. Analysis of promoter region of these differentially expressed orthologous genes revealed the existence of cis-regulatory elements associated with the differentiated expression pattern of these genes in the two rice cultivars. Further comparison of these regions in global rice population indicated their fixation and close relationship with rice population divergence., Conclusion: We proposed that variation in the expression patterns of these orthologous genes mediated by cis-regulatory elements in the two rice cultivars, may constitute an alternative evolutionary mechanism that distinguishes these two genetically and ecologically divergent rice cultivars in response to M. oryzae infection.

Fitness cost is a common phenomenon in rice blast disease-resistance breeding. MiR396 is a highly conserved microRNA (miRNA) family targeting Growth Regulating Factor ( OsGRF ) genes. Mutation at the target site of miR396 in certain OsGRF gene or blocking miR396 expression leads to increased grain yield. Here we demonstrated that fitness cost can be trade-off in miR396- OsGRF s module via balancing growth and immunity against the blast fungus. The accumulation of miR396 isoforms was significantly increased in a susceptible accession, but fluctuated in a resistant accession upon infection of Magnaporthe oryzae . The transgenic lines over-expressing different miR396 isoforms were highly susceptible to M. oryzae . In contrast, overexpressing target mimicry of miR396 to block its function led to enhanced resistance to M. oryzae in addition to improved yield traits. Moreover, transgenic plants overexpressing OsGRF6 , OsGRF7 , OsGRF8 , and OsGRF9 exhibited enhanced resistance to M. oryzae , but showed different alteration of growth. While overexpression of OsGRF7 led to defects in growth, overexpression of OsGRF6 , OsGRF8 , and OsGRF9 resulted in better or no significant change of yield traits. Collectively, our results indicate that miR396 negatively regulates rice blast disease- resistance via suppressing multiple OsGRF s, which in turn differentially control growth and yield. Therefore, miR396- OsGRFs could be a potential module to demolish fitness cost in rice blast disease-resistance breeding.

Blast disease of rice plant is caused by Magnaporthe oryzae (anamorph Pyricularia oryzae ). This disease is recognized to be one of the most serious diseases of rice crop around the world. A total of 72 monoconidial isolates of M. oryzae obtained from blast disease samples collected around Southern Karnataka were characterized using internal transcribed spacers of the ribosomal DNA sequences. These were analyzed by comparing with already deposited sequences in GenBank database. It helped in diagnosing the invasive pathogen in all locations. Variability of rDNA sequences was found to be highly polymorphic with 0.068962 nucleotide diversity showing 6 distinct clades. 33 haplotype groups were identified with haplotype diversity of 0.8881 and Tajima's neutrality test with a D value of -1.96827 with P < 0.05 showing the presence of variations among the sequences of pathogen isolates. The Tajima's D value of less than one indicates the presence of a high number of rare alleles. Our study indicates that the pathogen might have undergone recent selection pressure because of the exposure to a large number of cultivars resulting in the evolution of rare alleles. This shows the importance of characterizing internal transcribed spacer (ITS) to know pathogen diversity and its fitness which has potential to contribute to the field of breeding for blast disease resistance.

Background: Rice blast (caused by Magnaporthe oryzae) is one of the most destructive diseases of rice. While many blast resistance (R) genes have been identified and deployed in rice cultivars, little is known about the R gene-mediated defense mechanism. We used a rice transgenic line harboring the resistance gene Piz-t to investigate the R gene-mediated resistance response to infection., Results: We conducted comparative proteome profiling of the Piz-t transgenic Nipponbare line (NPB-Piz-t) and wild-type Nipponbare (NPB) inoculated with M. oryzae at 24, 48, 72 h post-inoculation (hpi) using isobaric tags for relative and absolute quantification (iTRAQ) analysis. Comparative analysis of the response of NPB-Piz-t to the avirulent isolate KJ201 and the virulent isolate RB22 identified 114 differentially expressed proteins (DEPs) between KJ201-inoculated NPB-Piz-t (KJ201-Piz-t) and mock-treated NPB-Piz-t (Mock-Piz-t), and 118 DEPs between RB22-inoculated NPB-Piz-t (RB22-Piz-t) and Mock-Piz-t. Among the DEPs, 56 occurred commonly in comparisons KJ201-Piz-t/Mock-Piz-t and RB22-Piz-t/Mock-Piz-t. In a comparison of the responses of NPB and NPB-Piz-t to isolate KJ201, 93 DEPs between KJ201-Piz-t and KJ201-NPB were identified. DEPs in comparisons KJ201-Piz-t/Mock-Piz-t, RB22-Piz-t/Mock-Piz-t and KJ201-Piz-t/KJ201-NPB contained a number of proteins that may be involved in rice response to pathogens, including pathogenesis-related (PR) proteins, hormonal regulation-related proteins, defense and stress response-related proteins, receptor-like kinase, and cytochrome P450. Comparative analysis further identified 7 common DEPs between the comparisons KJ201-Piz-t/KJ201-NPB and KJ201-Piz-t/RB22-Piz-t, including alcohol dehydrogenase I, receptor-like protein kinase, endochitinase, similar to rubisco large subunit, NADP-dependent malic enzyme, and two hypothetical proteins., Conclusions: Our results provide a valuable resource for discovery of complex protein networks involved in the resistance response of rice to blast fungus.

Rice blast, caused by the ascomycete Magnaporthe oryzae, is one of the most destructive rice diseases worldwide. Even though the disease has been present in California since 1996, there is no data for the pathogen population biology in the state. Using amplified fragment length polymorphisms and mating-type markers, the M. oryzae population diversity was investigated using isolates collected when the disease was first established in California and isolates collected a decade later. While in the 1990 samples, a single multilocus genotype (MLG) was identified (MLG1), over a decade later, we found 14 additional MLGs in the 2000 isolates. Some of these MLGs were found to infect the only rice blast-resistant cultivar (M-208) available for commercial production in California. The same samples also had a significant decrease of MLG1. MLG1 was found infecting the resistant rice cultivar M-208 on one occasion whereas MLG7 was the most common genotype infecting the M-208. MLG7 was identified in the 2000 samples, and it was not present in the M. oryzae population a decade earlier. Our results demonstrate a significant increase in genotypic diversity over time with no evidence of sexual reproduction and suggest a recent introduction of new virulent race(s) of the pathogen. In addition, our data could provide information regarding the durability of the Pi-z resistance gene of the M-208. This information will be critical to plant breeders in developing strategies for deployment of other rice blast resistance genes/cultivars in the future.

The emergence of new strains of Magnaporthe oryzae ( M. oryzae ) is associated with recurrent failure of resistance response mediated by single resistance ( R ) gene in rice. Therefore, stacking or combining of multiple R genes could improve the durability of resistance against multiple strains of M. oryzae . To achieve this, in the present study, intragenic stacking of rice blast resistance orthologue genes Pi54 and Pi54rh was performed through co-transformation approach. Both these genes were expressed under the control of independent promoters and blast susceptible indica rice line IET17021 was used for transformation. The highly virulent M. oryzae strain Mo-ei-ger1 that could knock down most of the major single blast R genes including Pi54 and exhibiting 89% virulence spectrum was used for phenotypic analysis. The stacked transgenic IET17021 lines ( Pi54  +  Pi54rh ) have shown complete resistance to Mo-ei-ger1 strain in comparison to non-transgenic lines. These two R gene stacked indica transgenic lines could serves as a novel germplasm for rice blast resistance breeding programmes., Competing Interests: Compliance with ethical standardsAll the authors declare no conflict of interest.

Evidently, epigenetics is at forefront in explaining the mechanisms underlying the success of human pathogens and in the identification of pathogen-induced modifications within host plants. However, there is a lack of studies highlighting the role of epigenetics in the modulation of the growth and pathogenicity of fungal plant pathogens. In this review, we attempt to highlight and discuss the role of epigenetics in the regulation of the growth and pathogenicity of fungal phytopathogens using Magnaporthe oryzae, a devastating fungal plant pathogen, as a model system. With the perspective of wide application in the understanding of the development, pathogenesis and control of other fungal pathogens, we attempt to provide a synthesized view of the epigenetic studies conducted on M. oryzae to date. First, we discuss the mechanisms of epigenetic modifications in M. oryzae and their impact on fungal development and pathogenicity. Second, we highlight the unexplored epigenetic mechanisms and areas of research that should be considered in the near future to construct a holistic view of epigenetic functioning in M. oryzae and other fungal plant pathogens. Importantly, the development of a complete understanding of the modulation of epigenetic regulation in fungal pathogens can help in the identification of target points to combat fungal pathogenesis., (© 2016 BSPP AND JOHN WILEY & SONS LTD.)

Rice blast disease is one of the most destructive rice diseases worldwide. The pi21 gene confers partial and durable resistance to Magnaporthe oryzae . However, little is known regarding the molecular mechanisms of resistance mediated by the loss-of-function of Pi21 . In this study, comparative transcriptome profiling of the Pi21 -RNAi transgenic rice line and Nipponbare with M. oryzae infection at different time points (0, 12, 24, 48, and 72 hpi) were investigated using RNA sequencing. The results generated 43,222 unique genes mapped to the rice genome. In total, 1109 differentially expressed genes (DEGs) were identified between the Pi21 -RNAi line and Nipponbare with M. oryzae infection, with 103, 281, 209, 69, and 678 DEGs at 0, 12, 24, 48, and 72 hpi, respectively. Functional analysis showed that most of the DEGs were involved in metabolism, transport, signaling, and defense. Among the genes assigned to plant-pathogen interaction, we identified 43 receptor kinase genes associated with pathogen-associated molecular pattern recognition and calcium ion influx. The expression levels of brassinolide-insensitive 1, flagellin sensitive 2, and elongation factor Tu receptor, ethylene (ET) biosynthesis and signaling genes, were higher in the Pi21 -RNAi line than Nipponbare. This suggested that there was a more robust PTI response in Pi21 -RNAi plants and that ET signaling was important to rice blast resistance. We also identified 53 transcription factor genes, including WRKY, NAC, DOF, and ERF families that show differential expression between the two genotypes. This study highlights possible candidate genes that may serve a function in the partial rice blast resistance mediated by the loss-of-function of Pi21 and increase our understanding of the molecular mechanisms involved in partial resistance against M. oryzae .

To identify a novel biopesticide controlling rice blast disease caused by Magnaporthe oryzae, 700 plant extracts were evaluated for their inhibitory effects on mycelial growth of M. oryzae. The L. foenum-graecum Herba extract showed the lowest inhibition concentration (IC50) of 39.28 μg/ml, which is lower than the IC50 of blasticidin S (63.06 μg/ml), a conventional fungicide for rice blast disease. When treatments were combined, the IC50 of blasticidin S was dramatically reduced to 10.67 μg/ml. Since ABC transporter genes are involved in fungicide resistance of many organisms, we performed RT-PCR to investigate the transcriptional changes of 40 ABC transporter family genes of M. oryzae treated with the plant extract, blasticidin S, and tetrandrine, a recognized ABC transporter inhibitor. Four ABC transporter genes were prominently activated by blasticidin S treatment, but were suppressed by combinational treatment of blasticidin S with the plant extract, or with tetrandrine that didn't show cellular toxicity by itself in this study. Mycelial death was detected via confocal microscopy at 24 h after plant extract treatment. Finally, subsequent rice field study revealed that the plant extract had high control efficacy of 63.3% and should be considered a biopesticide for rice blast disease. These results showed that extract of L. foenum graecum Herba suppresses M. oryzae ABC transporter genes inducing mycelial death and therefore may be a potent novel biopesticide.

Rice is a staple and most important security food crop consumed by almost half of the world's population. More rice production is needed due to the rapid population growth in the world. Rice blast caused by the fungus, Magnaporthe oryzae is one of the most destructive diseases of this crop in different part of the world. Breakdown of blast resistance is the major cause of yield instability in several rice growing areas. There is a need to develop strategies providing long-lasting disease resistance against a broad spectrum of pathogens, giving protection for a long time over a broad geographic area, promising for sustainable rice production in the future. So far, molecular breeding approaches involving DNA markers, such as QTL mapping, marker-aided selection, gene pyramiding, allele mining and genetic transformation have been used to develop new resistant rice cultivars. Such techniques now are used as a low-cost, high-throughput alternative to conventional methods allowing rapid introgression of disease resistance genes into susceptible varieties as well as the incorporation of multiple genes into individual lines for more durable blast resistance. The paper briefly reviewed the progress of studies on this aspect to provide the interest information for rice disease resistance breeding. This review includes examples of how advanced molecular method have been used in breeding programs for improving blast resistance. New information and knowledge gained from previous research on the recent strategy and challenges towards improvement of blast disease such as pyramiding disease resistance gene for creating new rice varieties with high resistance against multiple diseases will undoubtedly provide new insights into the rice disease control.

Plants have evolved intracellular immune receptors to detect pathogen proteins known as effectors. How these immune receptors detect effectors remains poorly understood. Here we describe the structural basis for direct recognition of AVR-Pik, an effector from the rice blast pathogen, by the rice intracellular NLR immune receptor Pik. AVR-PikD binds a dimer of the Pikp-1 HMA integrated domain with nanomolar affinity. The crystal structure of the Pikp-HMA/AVR-PikD complex enabled design of mutations to alter protein interaction in yeast and in vitro, and perturb effector-mediated response both in a rice cultivar containing Pikp and upon expression of AVR-PikD and Pikp in the model plant Nicotiana benthamiana. These data reveal the molecular details of a recognition event, mediated by a novel integrated domain in an NLR, which initiates a plant immune response and resistance to rice blast disease. Such studies underpin novel opportunities for engineering disease resistance to plant pathogens in staple food crops.

Rice blast resistance ( R ) genes-mediated resistance response depends on various resistance-related genes involved in incompatible interactions. In this work, the expression profiles of innate rice immunity related genes were examined in the mediated resistance response of true/field resistance genes. Three sets of rice near-isogenic lines (NILs) were used: the resistant NILs carrying true resistance genes in the genetic background of the susceptible cultivar Nipponbare (NB), NB-Pib, NB-Pizt, NB-Pik and NB-Pita2; NILs bearing field resistance genes pi21 in the susceptible cultivar Aichiasahi (AA) AA-pi21, Kahei (KHR). The marker gene OsWRKY45 of salicylic acid (SA) signalling was upregulated in all tested cultivars. And, JAmyb (marker gene of jasmonic acid signalling) showed higher upregulation in the resistance lines with nucleotide-binding sites and leucine-rich repeat (NBS-LRR) R genes Pib , Pizt , Pik , Pita2 and Pikahei than in NB and KHS. SalT of abscisic acid (ABA) signalling may be involved in the R/Avr interaction, including Pizt , Pik , pi21 and Pikahei . However, SalT was shown to negatively regulate Pib/AvrPib interaction. OsPR1b and PBZ1 were differentially expressed and strongly activated at a later stage by 48 h post-inoculation. Interestingly, there was evidence that OsPR1b and PBZ1 played an important role in the pi21- mediated response. It was shown that OsRAR1 could be upregulated in the true resistance line NB-Pita2 and the field resistance line KHR, while OsSGT1 and OsHSP90 could be upregulated in all tested lines. The involvement of these genes illustrated the complexity of the downstream signalling pathways in the mediated resistance response of true/field resistance genes.

Frequent and devastating epidemics of parasites are one of the major issues encountered by modern agriculture. To manage the impact of pathogens, resistant plant varieties have been selected. However, resistances are overcome by parasites requiring the use of pesticides and causing new economical and food safety issues. A promising strategy to maintain the epidemic at a low level and hamper pathogen's adaptation to varietal resistance is the use of mixtures of varieties such that the mix will form a heterogeneous environment for the parasite. A way to find the good combination of varieties that will actually constitute a heterogeneous environment for pathogens is to look for genotype × genotype (G × G) interactions between pathogens and plant varieties. A pattern in which pathogens have a high fitness on one variety and a poor fitness on other varieties guarantees the efficiency of the mixture strategy. In the present article, we inoculated 18 different genotypes of the fungus Magnaporthe oryzae on three rice plant varieties showing different levels of partial resistance in order to find a variety combination compatible with the requirements of the variety mixture strategy, i.e., showing appropriate G × G interactions. We estimated the success of each plant-fungus interaction by measuring fungal fitness and three fungal life history traits: infection success, within-host growth, sporulation capacity. Our results show the existence of G × G interactions between the two varieties Ariete and CO39 on all measured traits and fungal fitness. We also observed that these varieties have different resistance mechanisms; Ariete is good at controlling infection success of the parasite but is not able to control its growth when inside the leaf, while CO39 shows the opposite pattern. We also found that Maratelli's resistance has been eroded. Finally, correlation analyses demonstrated that not all infectious traits are positively correlated.

Magnaporthe oryzae (M. oryzae) is a fungal pathogen and the causal agent of rice blast disease. Previous lipidomics analysis of M. oryzae demonstrated that trehalose, a carbohydrate common to various fungi and algae, is thought to be involved in the possible conversion of glycogen into triacylglycerides for energy, an important step in the pathogenesis of M. oryzae. A key enzyme responsible for trehalose synthesis is trehalose-6-phosphate synthase 1 (Tps1). Therefore, we modeled the structure of Tps1 and sought to screen a chemical database in silico for possible inhibitors of the enzyme. Based on homologous alignment and sequence analysis, we first modeled the structure of Tps1 to determine the potential active site of the enzyme and its conformation. Using this model, we then undertook a docking study to determine the potential interaction that would manifest between Tsp1 and potential chemical inhibitors. Of the 400,000 chemicals screened in the Molecular Libraries Small Molecule Repository, we identified 45 potential candidates. The best candidate (Compound 24789937) was chosen and subjected to various structural optimization techniques to improve the suitability of the potential chemical inhibitors at the docking site of Tps1. From these modified versions of Compound 24789937, one lead compound (Lead 25) was shown to have the best binding affinity to Tps1 and good water solubility as compared with the ideal template compound and the other 44 potential candidates. Molecular dynamics simulation further confirmed the strength of the Tps1-Lead 25 complex and indicated the potential for Lead 25 to be used as an inhibitor of Tps1 in the control of M. oryzae-mediated rice blast disease.