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.     

2,6‐Dimethoxy‐1,4‐Benzoquinone Enhances Resistance Against the Rice Blast Fungus Magnaporthe oryzae

We investigated the effect of 2,6‐dimethoxy‐1,4‐benzoquinone (DMBQ) on induced resistance to Magnaporthe oryzae in rice. DMBQ concentrations greater than 50 μg/ml inhibited spore germination and appressorium formation in M. oryzae. When rice leaves pretreated with 10 μg/ml DMBQ, which did not show antifungal activity against spore germination and appressorium formation of M. oryzae, were inoculated with M. oryzae spores 5 days after DMBQ pretreatment, blast lesion formation was inhibited compared with control leaves pretreated with distilled water. In addition, infection‐inhibiting activity against M. oryzae was significantly enhanced in rice leaf sheaths pretreated with 10 μg/ml DMBQ. H₂O₂ generation was observed in rice leaves pretreated with DMBQ, and PAL, POX, CHS and PR10a were significantly expressed in these leaves. These results suggested that DMBQ can protect rice from blast disease caused by M. oryzae.     

MoWhi2 regulates appressorium formation and pathogenicity via the MoTor signalling pathway in Magnaporthe oryzae

Magnaporthe oryzae causes rice blast disease, which seriously threatens the safety of food production. Understanding the mechanism of appressorium formation, which is one of the key steps for successful infection by M. oryzae, is helpful to formulate effective control strategies of rice blast. In this study, we identified MoWhi2, the homolog of Saccharomyces cerevisiae Whi2 (Whisky2), as an important regulator that controls appressorium formation in M. oryzae. When MoWHI2 was disrupted, multiple appressoria were formed by one conidium and pathogenicity was significantly reduced. A putative phosphatase, MoPsr1, was identified to interact with MoWhi2 using a yeast two-hybridization screening assay. The knockout mutant ΔMopsr1 displayed similar phenotypes to the ΔMowhi2 strain. Both the ΔMowhi2 and ΔMopsr1 mutants could form appressoria on a hydrophilic surface with cAMP levels increasing in comparison with the wild type (WT). The conidia of ΔMowhi2 and ΔMopsr1 formed a single appressorium per conidium, similar to WT, when the target of rapamycin (TOR) inhibitor rapamycin was present. In addition, compared with WT, the expression levels of MoTOR and the MoTor signalling activation marker gene MoRS3 were increased, suggesting that inappropriate activation of the MoTor signalling pathway is one of the important reasons for the defects in appressorium formation in the ΔMowhi2 and ΔMopsr1 strains. Our results provide insights into MoWhi2 and MoPsr1-mediated appressorium development and pathogenicity by regulating cAMP levels and the activation of MoTor signalling in M. oryzae.     

SSRs for Marker-Assisted Selection for Blast Resistance in Rice (Oryza sativa L.)

Rice blast caused by the fungus Magnaporthe oryzae is one of the most devastating diseases of rice in nearly all rice growing areas of the world including Malaysia. To develop cultivars with resistance against different races of M. oryzae, availability of molecular markers along with marker-assisted selection strategies are essential. In this study, 11 polymorphic simple sequence repeat (SSR) markers with good fit of 1:2:1 ratio for single gene model in F₂ population derived from the cross of Pongsu seribu 2 (Resistant) and Mahsuri (Susceptible) rice cultivars were analysed in 296 F₃ families derived from individual F₂ plants to investigate association with Pi gene conferring resistance to M. oryzae pathotype. Parents and progeny were grouped into two phenotypic classes based on their blast reactions. Chi-square test for the segregation of resistance and susceptibility in F₃ generation fitted a ratio of approximately 3:1. Association of SSR markers with phenotypic trait in F₃ families was identified by statistical analysis. Four SSR markers (RM413, RM5961, RM1233 and RM8225) were significantly associated with blast resistance to pathotype 7.2 of M. oryzae in rice (p ≤ 0.01). These four markers accounted for about 20% of total phenotypic variation. So, these markers were confirmed as suitable markers for use in marker-assisted selection and confirmation of blast resistance genes to develop rice cultivars with durable blast resistance in Malaysian rice breeding programmes.     

Identification of the blast resistance gene Picl(t) from Chaling common wild rice (Oryza rufipogon Griff.)

Rice blast, caused by the fungal pathogen Magnaporthe oryzae (M. oryzae), is one of the most destructive and widespread plant diseases in the world. Utilization of resistance genes in rice breeding is considered to be an effective and economical method to control this disease. To identify new sources of blast resistance, a set of 1160 introgression lines (ILs) containing chromosome segments of Chaling common wild rice (Oryza rufipogon Griff.) in the genetic background of an elite indica rice variety 93-11 were developed and phenotyped in the blast nursery. Thirty-three ILs displaying stable blast resistance in three consecutive years were obtained. Among them, one line, IL1043, was subsequently found to be resistant to all of the 28 M. oryzae isolates from different regions through artificial inoculation in greenhouse. By combining bulk segregant analysis coupled with next-generation sequencing (BSA-seq) and recessive class analysis (RCA), a major blast resistance gene in IL1043, designated Picl(t), was mapped on rice chromosome 6 flanked by the markers RM527 and Indel6 with an interval of approximately 925 kb, which covers the Pi2/9 locus. These results will facilitate fine mapping and cloning of Picl(t), and the linked markers will further provide a useful tool for rice blast resistance breeding.     

Introgression of Pi2 and Pi5 Genes for Blast (Magnaporthe oryzae) Resistance in Rice and Field Evaluation of Introgression Lines for Resistance and Yield Traits

Blast caused by Magnaporthe oryzae is the most devastating disease causing significant loss in rice production. The destructive nature of the disease is mainly due to the genetic plasticity of M. oryzae which complicates the breeding strategies. Blast can be effectively managed by the deployment of R genes. In this study, broad‐spectrum blast resistance genes Pi2 and Pi5 were introgressed independently into popular but blast susceptible rice variety, Samba Mahsuri (BPT5204) by applying marker‐assisted backcross breeding approach. Tightly linked markers AP5930 for Pi2 and 40N23r for Pi5 gene were used in foreground selection. Background selection helped to identify the lines with maximum recovery of recurrent parent genome (RPG). The RPG recovery in Pi2 introgression lines was up to 90.17 and 91.46% in Pi5 lines. Homozygous introgression lines in BC₃F₄ generation carrying Pi2 and Pi5 gene were field evaluated for blast resistance, yield per se and yield‐related traits. The lines showed resistance to leaf and neck blast in multilocation field evaluation. Improved BPT5204 lines with improvement for blast resistance were on par with original BPT5204 in terms of grain yield and grain features.     

Identification of Sternbin and Naringenin as Detoxified Metabolites from the Rice Flavanone Phytoalexin Sakuranetin by Pyricularia oryzae

Sakuranetin ( 1 ) is a flavanone phytoalexin that has been reported to play an important role in disease resistance in rice plants. The rice blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae) has been reported to metabolize 1 to lower its antifungal activity. Here, two flavanones, sternbin ( 2 ) and naringenin ( 3 ), were identified as metabolites of 1 in P. oryzae suspension culture by liquid chromatography tandem mass spectrometry (LC/MS/MS). The inhibition of 1 , 2 , and 3 on P. oryzae mycelial growth were 45%, 19%, and 19%, respectively, at a concentration of 100 μm . Thus, 2 and 3 are detoxified metabolites of 1 by P. oryzae.     

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.     

Picolinic acid spray stimulates the antioxidative metabolism and minimizes impairments on photosynthesis on wheat leaves infected by Pyricularia oryzae

Fungal pathogens produce toxins that are important for their pathogenesis and/or aggressiveness towards their hosts. Picolinic acid (PA), a non‐host selective toxin, causes lesions on rice leaves resembling those originated from Pyricularia oryzae infection. Considering that non‐host selective toxins can be useful for plant diseases control, this study investigated whether the foliar spray with PA on wheat (Triticum aestivum L.) plants, in a non‐phytotoxic concentration, could increase their resistance to blast, stimulate the anti‐oxidative metabolism, and minimize alterations in photosynthesis. The PA spray at concentrations greater than 0.1 mg ml^?1 caused foliar lesions, compromised the photosynthesis and was linked with greater accumulation of hydrogen peroxide (H₂O₂) and superoxide anion radical (O₂^??). Fungal mycelial growth, conidia production and germination decreased by PA at 0.3 mg ml^?1. Blast severity was significantly reduced by 59 and 23%, respectively, at 72 and 96 h after inoculation for plants sprayed with PA (0.1 mg ml^?1) at 24 h before fungal inoculation compared to non‐sprayed plants. Reduction on blast symptoms was linked with increases on ascorbate peroxidase (EC 1.11.1.11), catalase (EC 1.11.1.6), glutathione peroxidase (EC 1.11.1.9), glutathione reductase (EC 1.8.1.7), glutathione‐S‐transferase (EC 2.5.1.18), peroxidase (EC 1.11.1.7), and superoxide dismutase (EC 1.15.1.1) activities, lower H₂O₂ and O₂^?? accumulation, reduced malondialdehyde production as well as less impairments to the photosynthetic apparatus. A more efficient antioxidative metabolism that rapidly scavenges the reactive oxygen species generated during P. oryzae infection, without dramatically decreasing the photosynthetic performance, was a remarkable effect obtained with PA spray.     

Intercellular Production of Tamavidin 1, a Biotin-Binding Protein from Tamogitake Mushroom,Confers Resistance to the Blast Fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> in Transgenic Rice

The blast fungus Magnaporthe oryzae, one of the most devastating rice pathogens in the world, shows biotin-dependent growth. We have developed a strategy for creating disease resistance to M. oryzae whereby intercellular production of tamavidin 1, a biotin-binding protein from Pleurotus cornucopiae occurs in transgenic rice plants. The gene that encodes tamavidin 1, fused to the sequence for a secretion signal peptide derived from rice chitinase gene, was connected to the Cauliflower mosaic virus 35S promoter, and the resultant construct was introduced into rice. The tamavidin 1 was accumulated at levels of 0.1–0.2% of total soluble leaf proteins in the transgenic rice and it was localized in the intercellular space of rice leaves. The tamavidin 1 purified from the transgenic rice was active, it bound to biotin and inhibited in vitro growth of M. oryzae by causing biotin deficiency. The transgenic rice plants showed a significant resistance to M. oryzae. This study shows the possibility of a new strategy to engineer disease resistance in higher plants by taking advantage of a pathogen’s auxotrophy.     

Inhibitory Action of Biofungicide Physcion on Initial and Secondary Infection of Magnaporthe oryzae

Rice blast, caused by the ascomycete fungus Magnaporthe oryzae, is one of the most destructive plant diseases in the world and a serious threat to global food security. Biofungicide physcion, extracted from roots of Chinese traditional herb rhubarb, has been commercialized to control cucumber powdery mildew (Podosphaera fuliginea) in China as a new active and environmentally‐friendly ingredient. This study investigated its bioactivity on rice blast and impact on the infection of M. oryzae. Physcion was effective on M. oryzae with 50% inhibition concentration of 18.19 mg/l on the fungus mycelium growth in vitro and provided 86 and 98% control efficacy in vivo at the concentrations of 40 and 60 mg/l, respectively. It inhibited the initial infection of M. oryzae by inhibiting growth of mycelia, the germination of conidia and the formation of appressoria. Moreover, it inhibited the secondary infection of the fungus at the concentration of 40 mg/l, but had no influence at the low concentrations of 1.25–20 mg/l. So physcion might be a promising natural plant extract that is worthy of being explored to apply on the control of rice blast.     

Isolation,fine mapping and expression profiling of a lesion mimic genotype, <Emphasis Type="Italic">spl</Emphasis><Superscript><Emphasis Type="Italic">NF4050</Emphasis>-<Emphasis Type="Italic">8</Emphasis></Superscript> that confers blast resistance in rice

We evaluated a large collection of Tos17 mutant panel lines for their reaction to three different races of Magnaporthe oryzae and identified a lesion mimic mutant, NF4050-8, that showed lesions similar to naturally occurring spl5 mutant and enhanced resistance to all the three blast races tested. Nested modified-AFLP using Tos17-specific primers and southern hybridization experiments of segregating individuals indicated that the lesion mimic phenotype in NF4050-8 is most likely due to a nucleotide change acquired during the culturing process and not due to Tos17 insertion per se. Inheritance and genetic analyses in two japonica × indica populations identified an overlapping genomic region of 13 cM on short arm of chromosome 7 that was linked with the lesion mimic phenotype. High-resolution genetic mapping using 950 F₃ and 3,821 F₄ plants of NF4050-8 × CO39 delimited a 35 kb region flanked by NBARC1 (5.262 Mb) and RM8262 (5.297 Mb), which contained 6 ORFs; 3 of them were ‘resistance gene related’ with typical NBS–LRR signatures. One of them harbored a NB–ARC domain, which had been previously demonstrated to be associated with cell death in animals. Microarray analysis of NF4050-8 revealed significant up-regulation of numerous defense/pathogenesis-related genes and down-regulation of heme peroxidase genes. Real-time PCR analysis of WRKY45 and PR1b genes suggested possible constitutive activation of a defense signaling pathway downstream of salicylic acid but independent of NH1 in these mutant lines of rice.     

Activation of Systemic Resistance to Magnaporthe oryzae in Rice by Substances Produced by Fusarium solani Isolated from Soil

Of 70 micro‐organisms (fungi, bacteria and actinomycetes) isolated from soil using vegetable tissue baits, 16 produced substances in culture fluids capable of preventing the development of blast caused by Magnaporthe oryzae on rice leaves with little or no inhibitory effect on the conidial germination of the pathogen. Isolate KS‐F14, which secreted substances capable of activating resistance in untreated leaves, was selected and identified as Fusarium solani. The resistance‐inducing substances were effective at pH values ranging from 5 to 10 and were stable under high temperatures, maintaining approximately the same level of activity even after autoclaving for 20 min. After application, the activated resistance in rice leaves persisted for 14 days. The polar solvent extracts of freeze‐dried KS‐F14 secretions were effective in activating resistance against M. oryzae in rice plants. The non‐polar solvent extracts were also effective, albeit not as effective as the polar solvent extracts, indicating that although the majority of the secreted resistance‐inducing compounds are hydrophilic, some of the compounds are hydrophobic. Treating secretions with cation or anion exchange resins only partially reduced their resistance‐inducing ability, suggesting that the resistance‐inducing components include both charged and non‐charged compounds. The resistance‐inducing compounds produced by F. solani have the potential to be developed into a commercial product for the control of rice blast and possibly other plant diseases.     

Silicon enhances photochemical efficiency and adjusts mineral nutrient absorption in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> infected rice plants

Silicon (Si) has been verified to play an important role in enhancing plant resistance against pathogens, but the exact mechanisms remain unclear. Two near-isogenic lines of rice (Oryza sativa L.), CO39 (blast susceptible), and C101LAC (Pi-1) (blast resistant), were hydroponically grown to study the effects of exogenous silicon application on the changes of disease incidence, mineral nutrient concentrations, chlorophyll content, and photochemical efficiency in Magnaporthe oryzae infected rice plants. Si amendment in nutrient solution at a concentration of 2.0 mM significantly reduced the disease index of rice plants of CO39 and C101LAC (Pi-1). Silicon application alone had no effects on mineral nutrient contents, chlorophyll content, maximum/potential quantum efficiency (F _v/F _m), and the maximum primary yield (F _v/F ₀) of photochemistry of PS II in healthy rice leaves. M. oryzae inoculation significantly increased the content of K, Na, Ca, Mg, Fe, and reduced the value of F _v/F ₀ and F _v/F _m in rice leaves. However, Si treatment suppressed M. oryzae induced increase of mineral nutrient contents, and significantly increased F _v/F ₀ and F _v/F _m value compared with Si-deficient infected plants. These results suggest that silicon-enhanced resistance to rice blast is associated with an enhancement of photochemical efficiency and adjustment of mineral nutrient absorption in M. oryzae-infected rice plants.     

Microbial secondary metabolite induction of abnormal appressoria formation mediates control of rice blast disease caused by Magnaporthe oryzae

Okinawa, the only subtropical area in Japan with numerous island ecosystems, is expected to have diverse microbial resources. Recently, we reported the construction of a culture filtrate library with microbes originally isolated from soils in Okinawa, including the Yaeyama Archipelago, and validated its phylogenetic diversity. In the present study, we investigated the inhibitory effect of the cell extract (CE) from microbial isolate 3–45 against Magnaporthe oryzae in rice (Oryza sativa). Abnormal appressorium formation by M. oryzae was induced in the presence of the CE from isolate 3–45. Additionally, melanization of appressoria was inhibited in the presence of CE from isolate 3–45. Sequence analysis of the 16S rDNA region of isolate 3–45 indicated that it shared similarities with Streptomyces erythrochromogenes. When rice leaves were inoculated with M. oryzae in the presence of CE from isolate 3–45, blast lesion formation was inhibited compared to leaves treated with M. oryzae in the absence of CE from isolate 3–45. In addition, M. oryzae infective activity was significantly inhibited in rice leaf sheaths treated with CE from isolate 3–45. Furthermore, abnormal appressorium formation was observed in the presence of heat‐treated CE from isolate 3–45. These results suggest that CE from isolate 3–45 can protect rice from blast disease caused by M. oryzae. Further studies are required to identify the active compounds present in 3–45‐CE and to clarify its mechanism of inhibition in full detail. The present study on isolate 3–45 might contribute to the development of a new fungicide for controlling rice blast disease caused by M. oryzae.     

Proteomic analysis of rice mutants susceptible to Magnaporthe oryzae

To identify genes involved in rice Pi5-mediated disease resistance to Magnaporthe oryzae, we compared the proteomes of the RIL260 rice strain carrying the Pi5 resistance gene with its susceptible mutants M5465 and M7023. Proteins were extracted from the leaf tissues of both RIL260 and the mutant lines at 0, 24, and 48 h after M. oryzae inoculation and separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified eight proteins that were differently expressed between the resistant and susceptible plants (three down- and five up-regulated proteins in the mutants). The down-regulated proteins included a triosephosphate isomerase (spot no. 2210), a 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (no. 3611), and an unknown protein (no. 4505). In addition, the five up-regulated proteins in the mutants were predicted to be a fructokinase I (no. 313), a glutathione S-transferase (no. 2310), an atpB of chloroplast ATP synthase (no. 3616), an aminopeptidase N (no. 3724), and an unknown protein (no. 308). These results suggest that proteomic analysis of rice susceptible mutants is a useful method for identifying novel proteins involved in resistance to the M. oryzae pathogen.     

Deubiquitinase Ubp3 regulates ribophagy and deubiquitinates Smo1 for appressorium-mediated infection by Magnaporthe oryzae

The Ubp family of deubiquitinating enzymes has been found to play important roles in plant-pathogenic fungi, but their regulatory mechanisms are still largely unknown. In this study, we revealed the regulatory mechanism of the deubiquitinating enzyme Ubp3 during the infection process of Magnaporthe oryzae. AUBP3 deletion mutant was severely defective in appressorium turgor accumulation, leading to the impairment of appressorial penetration. During appressorium formation, the mutant was also defective in glycogen and lipid metabolism. Interestingly, we found that nitrogen starvation and rapamycin treatment induced the ribophagy process in M. oryzae, which is closely dependent on Ubp3. In the ∆ubp3 mutant, the ribosome proteins and rRNAs were not well degraded on nitrogen starvation and rapamycin treatment. We also found that Ubp3 interacted with the GTPase-activating protein Smo1 and regulated its de-ubiquitination. Ubp3-dependent de-ubiquitination of Smo1 may be required for Smo1 to coordinate Ras signalling. Taken together, our results showed at least two roles of Ubp3 in M. oryzae: it regulates the ribophagy process and it regulates de-ubiquitination of GTPase-activating protein Smo1 for appressorium-mediated infection.