Analysis of the Relationship between Blast Resistance Genes and Disease Resistance of Rice Germplasm via Functional Molecular Markers

Rice blast disease is one of the most devastating diseases of rice (Oryza sativa L.) caused by the fungus Magnaporthe oryzae (M. oryzae), and neck blast is the most destructive phase of this illness. The underlying molecular mechanisms of rice blast resistance are not well known. Thus, we collected 150 rice varieties from different ecotypes in China and assessed the rice blast resistances under the natural conditions that favoured disease development in Jining, Shandong Province, China in 2017. Results showed that 92 (61.3%) and 58 (38.7%) rice varieties were resistant and susceptible to M. oryzae, respectively. Among the 150 rice varieties screened for the presence of 13 major blast resistance (R) genes against M. oryzae by using functional markers, 147 contained one to eight R genes. The relationship between R genes and disease response was discussed by analysing the phenotype and genotype of functional markers. The results showed that the rice blast resistance gene Pita was significantly correlated with rice blast resistance. Our results provided a basis for the further understanding of the distribution of 13 major R genes of rice blast in the germplasm resources of the tested rice varieties, and were meaningful for rice disease resistance breeding.     

Comparative Nuclear Proteomics Analysis Provides Insight into the Mechanism of Signaling and Immune Response to Blast Disease Caused by Magnaporthe oryzae in Rice

Modulation of plant immune system by extrinsic/intrinsic factors and host‐specific determinants fine‐tunes cellular components involving multiple organelles, particularly nucleus to mount resistance against pathogen attack. Rice blast, caused by hemibiotrophic fungus Magnaporthe oryzae, is one of the most devastating diseases that adversely affect rice productivity. However, the role of nuclear proteins and their regulation in response to M. oryzae remains unknown. Here, the nucleus‐associated immune pathways in blast‐resistant rice genotype are elucidated. Temporal analysis of nuclear proteome is carried out using 2‐DE coupled MS/MS analysis. A total of 140 immune responsive proteins are identified associated with nuclear reorganization, cell division, energy production/deprivation, signaling, and gene regulation. The proteome data are interrogated using correlation network analysis that identified significant functional modules pointing toward immune‐related coinciding processes through a common mechanism of remodeling and homeostasis. Novel clues regarding blast resistance include nucleus‐associated redox homeostasis and glycolytic enzyme–mediated chromatin organization which manipulates cell division and immunity. Taken together, the study herein provides evidence that the coordination of nuclear function and reprogramming of host translational machinery regulate resistance mechanism against blast disease.     

Recent progress and understanding of the molecular mechanisms of the rice–Magnaporthe oryzae interaction

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is the most devastating disease of rice and severely affects crop stability and sustainability worldwide. This disease has advanced to become one of the premier model fungal pathosystems for host—pathogen interactions because of the depth of comprehensive studies in both species using modern genetic, genomic, proteomic and bioinformatic approaches. Many fungal genes involved in pathogenicity and rice genes involved in effector recognition and defence responses have been identified over the past decade. Specifically, the cloning of a total of nine avirulence (Avr) genes in M. oryzae, 13 rice resistance (R) genes and two rice blast quantitative trait loci (QTLs) has provided new insights into the molecular basis of fungal and plant interactions. In this article, we consider the new findings on the structure and function of the recently cloned R and Avr genes, and provide perspectives for future research directions towards a better understanding of the molecular underpinnings of the rice–M. oryzae interaction.     

Magnaporthe oryzae nucleoside diphosphate kinase is required for metabolic homeostasis and redox-mediated host innate immunity suppression

The fungus Magnaporthe oryzae causes blast, the most devastating disease of cultivated rice. After penetrating the leaf cuticle, M. oryzae grows as a biotroph in intimate contact with living rice epidermal cells before necrotic lesions develop. Biotrophic growth requires maintaining metabolic homeostasis while suppressing plant defenses, but the metabolic connections and requirements involved are largely unknown. Here, we characterized the M. oryzae nucleoside diphosphate kinase-encoding gene NDK1 and discovered it was essential for facilitating biotrophic growth by suppressing the host oxidative burst—the first line of plant defense. NDK enzymes reversibly transfer phosphate groups from tri- to diphosphate nucleosides. Correspondingly, intracellular nucleotide pools were perturbed in M. oryzae strains lacking NDK1 through targeted gene deletion, compared to WT. This affected metabolic homeostasis: TCA, purine and pyrimidine intermediates, and oxidized NADP⁺, accumulated in Δndk1. cAMP and glutathione were depleted. ROS accumulated in Δndk1 hyphae. Functional appressoria developed on rice leaf sheath surfaces, but Δndk1 invasive hyphal growth was restricted and redox homeostasis was perturbed, resulting in unsuppressed host oxidative bursts that triggered immunity. We conclude Ndk1 modulates intracellular nucleotide pools to maintain redox balance via metabolic homeostasis, thus quenching the host oxidative burst and suppressing rice innate immunity during biotrophy.     

Pex14/17, a filamentous fungus‐specific peroxin,is required for the import of peroxisomal matrix proteins and full virulence of Magnaporthe oryzae

Peroxisomes are ubiquitous organelles in eukaryotic cells that fulfil a variety of biochemical functions. The biogenesis of peroxisomes requires a variety of proteins, named peroxins, which are encoded by PEX genes. Pex14/17 is a putative recently identified peroxin, specifically present in filamentous fungal species. Its function in peroxisomal biogenesis is still obscure and its roles in fungal pathogenicity have not yet been documented. Here, we demonstrate the contributions of Pex14/17 in the rice blast fungus Magnaporthe oryzae (Mopex14/17) to peroxisomal biogenesis and fungal pathogenicity by targeting gene replacement strategies. Mopex14/17 has properties of both Pex14 and Pex17 with regard to its protein sequence. Mopex14/17 is distributed at the peroxisomal membrane and is essential for efficient peroxisomal targeting of proteins containing peroxisomal targeting signal 1. MoPEX19 deletion leads to the cytoplasmic distribution of Mopex14/17, indicating that the peroxisomal import of Pex14/17 is dependent on Pex19. The knockout mutants of MoPEX14/17 show reduced fatty acid utilization, reactive oxygen species (ROS) degradation and cell wall integrity. Moreover, Δmopex14/17 mutants show delayed conidial generation and appressorial formation, and a reduction in appressorial turgor accumulation and penetration ability in host plants. These defects result in a significant reduction in the virulence of the mutant. These data indicate that MoPEX14/17 plays a crucial role in peroxisome biogenesis and contributes to fungal development and pathogenicity.     

Molecular progress on the mapping and cloning of functional genes for blast disease in rice (Oryza sativa L.): current status and future considerations

Rice blast disease, which is caused by the fungal pathogen Magnaporthe oryzae, is a recurring problem in all rice-growing regions of the world. The use of resistance (R) genes in rice improvement breeding programmes has been considered to be one of the best options for crop protection and blast management. Alternatively, quantitative resistance conferred by quantitative trait loci (QTLs) is also a valuable resource for the improvement of rice disease resistance. In the past, intensive efforts have been made to identify major R-genes as well as QTLs for blast disease using molecular techniques. A review of bibliographic references shows over 100 blast resistance genes and a larger number of QTLs (～500) that were mapped to the rice genome. Of the blast resistance genes, identified in different genotypes of rice, ～22 have been cloned and characterized at the molecular level. In this review, we have summarized the reported rice blast resistance genes and QTLs for utilization in future molecular breeding programmes to introgress high-degree resistance or to pyramid R-genes in commercial cultivars that are susceptible to M. oryzae. The goal of this review is to provide an overview of the significant studies in order to update our understanding of the molecular progress on rice and M. oryzae. This information will assist rice breeders to improve the resistance to rice blast using marker-assisted selection which continues to be a priority for rice-breeding programmes.     

Mitochondrial dynamics and mitophagy are necessary for proper invasive growth in rice blast

Magnaporthe oryzae causes blast disease, which is one of the most devastating infections in rice and several important cereal crops. Magnaporthe oryzae needs to coordinate gene regulation, morphological changes, nutrient acquisition and host evasion in order to invade and proliferate within the plant tissues. Thus far, the molecular mechanisms underlying the regulation of invasive growth in planta have remained largely unknown. We identified a precise filamentous-punctate-filamentous cycle in mitochondrial morphology during Magnaporthe–rice interaction. Interestingly, disruption of such mitochondrial dynamics by deletion of genes regulating either the mitochondrial fusion (MoFzo1) or fission (MoDnm1) machinery, or inhibition of mitochondrial fission using Mdivi-1 caused significant reduction in M. oryzae pathogenicity. Furthermore, exogenous carbon source(s) but not antioxidant treatment delayed such mitochondrial dynamics/transition during invasive growth. In contrast, carbon starvation induced the breakdown of the mitochondrial network and led to more punctate mitochondria in vitro. Such nutrient-based regulation of organellar dynamics preceded MoAtg24-mediated mitophagy, which was found to be essential for proper biotrophic development and invasive growth in planta. We propose that precise mitochondrial dynamics and mitophagy occur during the transition from biotrophy to necrotrophy and are required for proper induction and establishment of the blast disease in rice.     

Biochemical defence responses of resistant and susceptible rice genotypes against blast pathogen Magnaporthe oryzae

Abstract

Rice blast is the leading fungal disease which is caused by Magnaporthe oryzae that contributes for the significant decline in the rice yield throughout the globe. There is a need for the understanding of biochemical changes in rice plant during blast infection for the development of novel disease control strategies. In the present study, we isolated M. oryzae from the local paddy fields and the fungal isolates (VCF and PON) were identified by ITS-PCR using genomic DNA samples. Further, we inoculated resistant (BR2655 and TUNGA) and susceptible (INTAN and HR12) rice cultivars with PON and VCF isolates. PON isolate showed relatively high virulence compared to VCF and standard MTCC fungal strains. Therefore, we evaluated the effect of PON on the total protein content and plant defence-related key enzymes (peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, β-glucosidase, chitinase and lipoxygenase) activities between 24- and 120-hour post-inoculation (hpi). The results demonstrated the decrease in total protein content in all the inoculated cultivars. In addition, we observed the variation in the activity of peroxidase, polyphenol oxidase, β-glucosidase, chitinase and lipoxygenase at different time points in all the tested rice plants compared to respective controls. However, no significant difference was observed in the phenylalanine ammonia lyase activity relative to its control. Taken together, this study emphasizes on the variation in the activities of plant defence enzymes in different plant cultivars against the tested fungal pathogen and also implementation of defence enzymes as biochemical markers for resistant breeding.     

The DnaJ protein OsDjA6 negatively regulates rice innate immunity to the blast fungus Magnaporthe oryzae

Rice blast, caused by Magnaporthe oryzae (synonym: Pyricularia oryzae), severely reduces rice production and grain quality. The molecular mechanism of rice resistance to M. oryzae is not fully understood. In this study, we identified a chaperone DnaJ protein, OsDjA6, which is involved in basal resistance to M. oryzae in rice. The OsDjA6 protein is distributed in the entire rice cell. The expression of OsDjA6 is significantly induced in rice after infection with a compatible isolate. Silencing of OsDjA6 in transgenic rice enhances resistance to M. oryzae and also results in an increased burst of reactive oxygen species after flg22 and chitin treatments. In addition, the expression levels of WRKY45, NPR1 and PR5 are increased in OsDjA6 RNAi plants, indicating that OsDjA6 may mediate resistance by affecting the salicylic acid pathway. Finally, we found that OsDjA6 interacts directly with the E3 ligase OsZFP1 in vitro and in vivo. These results suggest that the DnaJ protein OsDjA6 negatively regulates rice innate immunity, probably via the ubiquitination proteasome degradation pathway.     

Sex at the origin: an Asian population of the rice blast fungus Magnaporthe oryzae reproduces sexually

Sexual reproduction may be cryptic or facultative in fungi and therefore difficult to detect. Magnaporthe oryzae, which causes blast, the most damaging fungal disease of rice, is thought to originate from southeast Asia. It reproduces asexually in all rice‐growing regions. Sexual reproduction has been suspected in limited areas of southeast Asia, but has never been demonstrated in contemporary populations. We characterized several M. oryzae populations worldwide both biologically and genetically, to identify candidate populations for sexual reproduction. The sexual cycle of M. oryzae requires two strains of opposite mating types, at least one of which is female‐fertile, to come into contact. In one Chinese population, the two mating types were found to be present at similar frequencies and almost all strains were female‐fertile. Compatible strains from this population completed the sexual cycle in vitro and produced viable progenies. Genotypic richness and linkage disequilibrium data also supported the existence of sexual reproduction in this population. We resampled this population the following year, and the data obtained confirmed the presence of all the biological and genetic characteristics of sexual reproduction. In particular, a considerable genetic reshuffling of alleles was observed between the 2 years. Computer simulations confirmed that the observed genetic characteristics were unlikely to have arisen in the absence of recombination. We therefore concluded that a contemporary population of M. oryzae, pathogenic on rice, reproduces sexually in natura in southeast Asia. Our findings provide evidence for the loss of sexual reproduction by a fungal plant pathogen outside its centre of origin.     

Two nucleotide sugar transporters are important for cell wall integrity and full virulence of Magnaporthe oryzae

Cell wall polysaccharides play key roles in fungal development, virulence, and resistance to the plant immune system, and are synthesized from many nucleotide sugars in the endoplasmic reticulum (ER)-Golgi secretory system. Nucleotide sugar transporters (NSTs) are responsible for transporting cytosolic-derived nucleotide sugars to the ER lumen for processing, but their roles in plant-pathogenic fungi remain to be revealed. Here, we identified two important NSTs, NST1 and NST2, in the rice blast fungus Magnaporthe oryzae. Both NSTs were localized in the ER, which was consistent with a function in transporting nucleotide sugar for processing in the ER. Sugar transport property analysis suggested that NST1 is involved in transportation of mannose and glucose, while NST2 is only responsible for mannose transportation. Accordingly, deletion of NSTs resulted in a significant decrease in corresponding soluble saccharides abundance and defect in sugar utilization. Moreover, both NSTs played important roles in cell wall integrity, were involved in asexual development, and were required for full virulence. The NST mutants exhibited decreasing external glycoproteins and exposure of inner chitin, which resulted in activation of the host defence response. Altogether, our results revealed that two sugar transporters are required for fungal cell wall polysaccharides accumulation and full virulence of M. oryzae.     

Twinfilin regulates actin assembly and Hexagonal peroxisome 1 (Hex1) localization in the pathogenesis of rice blast fungus Magnaporthe oryzae

Actin assembly at the hyphal tip is key for polar growth and pathogenesis of the rice blast fungus Magnaporthe oryzae. The mechanism of its precise assemblies and biological functions is not understood. Here, we characterized the role of M. oryzae Twinfilin (MoTwf) in M. oryzae infection through organizing the actin cables that connect to Spitzenkörper (Spk) at the hyphal tip. MoTwf could bind and bundle the actin filaments. It formed a complex with Myosin2 (MoMyo2) and the Woronin body protein Hexagonal peroxisome 1 (MoHex1). Enrichment of MoMyo2 and MoHex1 in the hyphal apical region was disrupted in a ΔMotwf loss-of-function mutant, which also showed a decrease in the number and width of actin cables. These findings indicate that MoTwf participates in the virulence of M. oryzae by organizing Spk-connected actin filaments and regulating MoHex1 distribution at the hyphal tip.     

Phosphate excess increases susceptibility to pathogen infection in rice

Phosphorus (P) is an essential nutrient for plant growth and productivity. Due to soil fixation, however, phosphorus availability in soil is rarely sufficient to sustain high crop yields. The overuse of fertilizers to circumvent the limited bioavailability of phosphate (Pi) has led to a scenario of excessive soil P in agricultural soils. Whereas adaptive responses to Pi deficiency have been deeply studied, less is known about how plants adapt to Pi excess and how Pi excess might affect disease resistance. We show that high Pi fertilization, and subsequent Pi accumulation, enhances susceptibility to infection by the fungal pathogen Magnaporthe oryzae in rice. This fungus is the causal agent of the blast disease, one of the most damaging diseases of cultivated rice worldwide. Equally, MIR399f overexpression causes an increase in Pi content in rice leaves, which results in enhanced susceptibility to M. oryzae. During pathogen infection, a weaker activation of defence-related genes occurs in rice plants over-accumulating Pi in leaves, which is in agreement with the phenotype of blast susceptibility observed in these plants. These data support that Pi, when in excess, compromises defence mechanisms in rice while demonstrating that miR399 functions as a negative regulator of rice immunity. The two signalling pathways, Pi signalling and defence signalling, must operate in a coordinated manner in controlling disease resistance. This information provides a basis to understand the molecular mechanisms involved in immunity in rice plants under high Pi fertilization, an aspect that should be considered in management of the rice blast disease.     

A Rapid and Simple Method for DNA Preparation of Magnaporthe oryzae from Single Rice Blast Lesions for PCR-Based Molecular Analysis

Rice blast is one of the most destructive diseases of rice worldwide, and the causative agent is the filamentous ascomycete Magnaporthe oryzae. With the successful cloning of more and more avirulence genes from M. oryzae, the direct extraction of M. oryzae genomic DNA from infected rice tissue would be useful alternative for rapid monitoring of changes of avirulence genes without isolation and cultivation of the pathogen. In this study, a fast, low-cost and reliable method for DNA preparation of M. oryzae from a small piece of infected single rice leaf or neck lesion was established. This single step method only required 10 min for DNA preparation and conventional chemical reagents commonly found in the laboratory. The AvrPik and AvrPi9 genes were successfully amplified with the prepared DNA. The expected DNA fragments from 570 bp to 1,139 bp could be amplified even three months after DNA preparation. This method was also suitable for DNA preparation from M. oryzae strains stored on the filter paper. All together these results indicate that the DNA preparation method established in this study is reliable, and could meet the basic needs for polymerase chain reaction-based analysis of M. oryzae.     

Expression of a bacterial chitosanase in rice plants improves disease resistance to the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Plant fungal pathogens change their cell wall components during the infection process to avoid degradation by host lytic enzymes, and conversion of the cell wall chitin to chitosan is likely to be one infection strategy of pathogens. Thus, introduction of chitosan-degradation activity into plants is expected to improve fungal disease resistance. Chitosanase has been found in bacteria and fungi, but not in higher plants. Here, we demonstrate that chitosanase, Cho1, from Bacillus circulans MH-K1 has antifungal activity against the rice blast fungus Magnaporthe oryzae. Introduction of the cho1 gene conferred chitosanase activity to rice cells. Transgenic rice plants expressing Cho1 designed to be localized in the apoplast showed increased resistance to M. oryzae accompanied by increased generation of hydrogen peroxide in the infected epidermal cells. These results strongly suggest that chitosan exists in the enzyme-accessible surface of M. oryzae during the infection process and that the enhancement of disease resistance is attributable to the antifungal activity of the secreted Cho1 and to increased elicitation of the host defense response.