Backbone and sidechain <Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N chemical shift assignments of the hydrophobin MPG1 from the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Fungal hydrophobins are secreted proteins that self-assemble at hydrophobic:hydrophilic interfaces. They are essential for a variety of processes in the fungal life cycle, including mediating interactions with surfaces and infection of hosts. The fungus Magnaporthe oryzae, the causative agent of rice blast, relies on the unique properties of hydrophobins to infect cultivated rice as well as over 50 different grass species. The hydrophobin MPG1 is highly expressed during rice blast pathogenesis and has been implicated during host infection. Here we report the backbone and sidechain assignments for the class I hydrophobin MPG1 from the rice blast fungus Magnaporthe oryzae.     

Rice blast fungus (Magnaporthe oryzae) infects Arabidopsis via a mechanism distinct from that required for the infection of rice

Magnaporthe oryzae is a hemibiotrophic fungal pathogen that causes rice (Oryza sativa) blast. Although M. oryzae as a whole infects a wide variety of monocotyledonous hosts, no dicotyledonous plant has been reported as a host. We found that two rice pathogenic strains of M. oryzae, KJ201 and 70-15, interacted differentially with 16 ecotypes of Arabidopsis (Arabidopsis thaliana). Strain KJ201 infected all ecotypes with varying degrees of virulence, whereas strain 70-15 caused no symptoms in certain ecotypes. In highly susceptible ecotypes, small chlorotic lesions appeared on infected leaves within 3 d after inoculation and subsequently expanded across the affected leaves. The fungus produced spores in susceptible ecotypes but not in resistant ecotypes. Fungal cultures recovered from necrotic lesions caused the same symptoms in healthy plants, satisfying Koch's postulates. Histochemical analyses showed that infection by the fungus caused an accumulation of reactive oxygen species and eventual cell death. Similar to the infection process in rice, the fungus differentiated to form appressorium and directly penetrated the leaf surface in Arabidopsis. However, the pathogenic mechanism in Arabidopsis appears distinct from that in rice; three fungal genes essential for pathogenicity in rice played only limited roles in causing disease symptoms in Arabidopsis, and the fungus seems to colonize Arabidopsis as a necrotroph through the secretion of phytotoxic compounds, including 9,12-octadecadienoic acid. Expression of PR-1 and PDF1.2 was induced in response to infection by the fungus, suggesting the activation of salicylic acid- and jasmonic acid/ethylene-dependent signaling pathways. However, the roles of these signaling pathways in defense against M. oryzae remain unclear. In combination with the wealth of genetic and genomic resources available for M. oryzae, this newly established pathosystem allows comparison of the molecular and cellular mechanisms underlying pathogenesis and host defense in two well-studied model plants.     

Sequence variation of avirulence gene AVR-Pita1 in rice blast fungus, Magnaporthe oryzae

The interaction between rice, Oryza sativa, and rice blast fungus, Magnaporthe oryzae, is triggered by an interaction between the protein products of the host resistant gene, and the pathogen avirulence gene. This interaction follows the ‘gene-for-gene' concept. The resistant gene has effectively protected rice plants from rice blast infection. However, the resistant genes usually break down several years after the release of the resistant rice varieties because the fungus has evolved to new races. The objective of this study is to investigate the nucleotide sequence variation of the AVR-Pita1 gene that influences the adaption of rice blast fungus to overcome the resistant gene, Pi-ta. Thirty rice blast fungus isolates were collected in 2005 and 2010 from infected rice plants in northern and northeastern Thailand. The nucleotide sequences of AVR-Pita1 were amplified and analyzed. Phylogenetic analysis was conducted using the MEGA 5.0 program. The results showed a high level of nucleotide sequence polymorphisms and the positive genetic selection pressure in Thai rice blast isolates. The details of sequence variation analysis were described in this article. The information from this study can be used for rice blast resistant breeding program in the future.     

Detection of novel blast resistance genes, Pi58(t) and Pi59(t), in a Myanmar rice landrace based on a standard differential system

The use of broad-spectrum R genes is an effective way to achieve durable resistance against rice blast (Magnaporthe oryzae Couch, anamorph: Pyricularia oryzae Cavara) in rice (Oryza sativa L.). We previously surveyed the diversity of blast resistance in 948 rice varieties and found a Myanmar rice landrace, Haoru (International Rice Research Institute genebank acc. no. IRGC33090), with broad-spectrum resistance against the standard differential blast isolates. Here, we examined the genetic basis of Haoru’s broad-spectrum resistance by using the standard blast differential system consisting of the standard isolates and differential varieties. For genetic analysis, we used a BC1F1 population and BC1F2 lines derived from crosses of Haoru with a susceptible variety, US-2. Co-segregation analysis of the reaction pattern in the BC1F1 population against the 20 standard isolates suggested that Haoru harbors three R genes. By using bulk-segregant and linkage analysis, we mapped two of the three R genes on chromosomes 12 and 6, and designated them as Pi58(t) and Pi59(t), respectively. Pi58(t) and Pi59(t) were differentiated from other reported R genes using the standard differential system. The estimated resistance spectrum of Pi58(t) corresponded with that of Haoru, suggesting that Pi58(t) is primarily responsible for Haoru’s broad-spectrum resistance. In addition, Pi59(t) and the third gene were also proven to be new and useful genetic resources for studying and improving blast resistance in rice.     

Interaction Transcriptome Analysis Identifies Magnaporthe oryzae BAS1-4 as Biotrophy-Associated Secreted Proteins in Rice Blast Disease

Biotrophic invasive hyphae (IH) of the blast fungus Magnaporthe oryzae secrete effectors to alter host defenses and cellular processes as they successively invade living rice (Oryza sativa) cells. However, few blast effectors have been identified. Indeed, understanding fungal and rice genes contributing to biotrophic invasion has been difficult because so few plant cells have encountered IH at the earliest infection stages. We developed a robust procedure for isolating infected-rice sheath RNAs in which ∼20% of the RNA originated from IH in first-invaded cells. We analyzed these IH RNAs relative to control mycelial RNAs using M. oryzae oligoarrays. With a 10-fold differential expression threshold, we identified known effector PWL2 and 58 candidate effectors. Four of these candidates were confirmed to be fungal biotrophy-associated secreted (BAS) proteins. Fluorescently labeled BAS proteins were secreted into rice cells in distinct patterns in compatible, but not in incompatible, interactions. BAS1 and BAS2 proteins preferentially accumulated in biotrophic interfacial complexes along with known avirulence effectors, BAS3 showed additional localization near cell wall crossing points, and BAS4 uniformly outlined growing IH. Analysis of the same infected-tissue RNAs with rice oligoarrays identified putative effector-induced rice susceptibility genes, which are highly enriched for sensor-transduction components rather than typically identified defense response genes.     

Herbivore-induced rice resistance against rice blast mediated by salicylic acid

In agro-ecosystems,plants are important mediators of interactions between their associated herbivorous insects and microbes,and any change in plants induced by one species may lead to cascading effects on interactions with other species.Often,such effects are regulated by phytohormones such as jasmonic acid(JA)and salicylic acid(SA).Here,we investigated the tripartite interactions among rice plants,three insect herbivores(Chilo suppressalis,Cnaphalocrocis medinalis or Nilapai-vata lugens),and the causal agent of rice blast disease,the fungus Magnaporthe oryzae.We found that pre-infestation of rice by C.suppressalis or N.lugens but not by C.medinalis conferred resistance to M.oryzae.For C.suppressalis and N.lugens,insect infestation without fungal inoculation induced the accumulation of both JA and SA in rice leaves.In contrast,infestation by C.medinalis increased JA levels but reduced SA levels.The exogenous application of SA but not of JA conferred resistance against M.oryzae.These results suggest that preinfestation by C suppressalis or N.lugens conferred resistance against M.oryzae by increasing SA accumulation.These findings enhance our understanding of the interactions among rice plant,insects and pathogens,and provide valuable information for developing an ecologically sound strategy for controlling rice blast.     

Differential Gene Expression Reflects Morphological Characteristics and Physiological Processes in Rice Immunity against Blast Pathogen Magnaporthe oryzae

The rice blast fungus Magnaporthe oryzae is a serious pathogen that jeopardises the world’s most important food-security crop. Ten common Malaysian rice varieties were examined for their morphological, physiological and genomic responses to this rice blast pathogen. qPCR quantification was used to assess the growth of the pathogen population in resistant and susceptible rice varieties. The chlorophyll content and photosynthesis were also measured to further understand the disruptive effects that M. oryzae has on infected plants of these varieties. Real-time PCR was used to explore the differential expression of eight blast resistance genes among the ten local varieties. Blast disease has destructive effects on the growth of rice, and the findings of our study provide evidence that the Pikh, Pi9, Pi21, and Osw45 genes are involved in defence responses in the leaves of Malaysian rice at 31 h after inoculation with M. oryzae pathotype P7.2. Both the chlorophyll content and photosynthesis were reduced, but the levels of Pikh gene expression remained constant in susceptible varieties, with a developed pathogen population and mild or severe symptoms. The Pi9, Pi21, and Osw45 genes, however, were simultaneously upregulated in infected rice plants. Therefore, the presence of the Pikh, Pi9, Pi21, and Osw45 genes in the germplasm is useful for improving the resistance of rice varieties.     

Evidence for Biotrophic Lifestyle and Biocontrol Potential of Dark Septate Endophyte Harpophora oryzae to Rice Blast Disease

The mutualism pattern of the dark septate endophyte (DSE) Harpophora oryzae in rice roots and its biocontrol potential in rice blast disease caused by Magnaporthe oryzae were investigated. Fluorescent protein-expressing H. oryzae was used to monitor the colonization pattern. Hyphae invaded from the epidermis to the inner cortex, but not into the root stele. Fungal colonization increased with root tissue maturation, showing no colonization in the meristematic zone, slight colonization in the elongation zone, and heavy colonization in the differentiation zone. H. oryzae adopted a biotrophic lifestyle in roots accompanied by programmed cell death. Real-time PCR facilitated the accurate quantification of fungal growth and the respective plant response. The biocontrol potential of H. oryzae was visualized by inoculation with eGFP-tagged M. oryzae in rice. H. oryzae protected rice from M. oryzae root invasion by the accumulation of H₂O₂ and elevated antioxidative capacity. H. oryzae also induced systemic resistance against rice blast. This systemic resistance was mediated by the OsWRKY45-dependent salicylic acid (SA) signaling pathway, as indicated by the strongly upregulated expression of OsWRKY45. The colonization pattern of H. oryzae was consistent with the typical characteristics of DSEs. H. oryzae enhanced local resistance by reactive oxygen species (ROS) and high antioxidative level and induced OsWRKY45-dependent SA-mediated systemic resistance against rice blast.     

Molecular breeding: marker-assisted selection combined with biolistic transformation for blast and bacterial blight resistance in Indica rice (cv. CO39)

Based on blast pathogen population dynamics and lineage exclusion assays, we found that the major blast resistance genes Pi-1 and Piz-5 confer resistance against most Magnaporthe grisea lineages. Near-isogenic rice lines C101LAC and C101A51 carrying these two major genes for blast resistance in the background of a most blast-susceptible genotype were used for developing the pyramids. The closely linked RFLP marker RZ536 and NBS-LRR r10 marker for Pi-1 and a PCR-based SAP marker RG64 for Piz-5 were used to identify the genes in the parents and in marker-assisted breeding of the pyramided populations. To achieve multiple resistance against blast and blight in this cultivar, these blast-resistant pyramids were transformed with the cloned bacterial blight resistance gene Xa21 known to confer resistance to all races of Xanthomonas oryzae pv. oryzae (Xoo). Bioassays with six independent transformants showed that transgenic CO39 plants were resistant to both pathogens, M. grisea and Xoo. We report here the stacking of three major genes (Pi-1 + Piz-5 + Xa21) into rice using two different approaches of molecular breeding: marker-assisted selection (MAS) and genetic transformation.     

Genome-wide association mapping of virulence gene in rice blast fungus Magnaporthe oryzae using a genotyping by sequencing approach

Magnaporthe oryzae is a fungal pathogen causing blast disease in many plant species. In this study, seventy three isolates of M. oryzae collected from rice (Oryza sativa) in 1996–2014 were genotyped using a genotyping-by-sequencing approach to detect genetic variation. An association study was performed to identify single nucleotide polymorphisms (SNPs) associated with virulence genes using 831 selected SNP and infection phenotypes on local and improved rice varieties. Population structure analysis revealed eight subpopulations. The division into eight groups was not related to the degree of virulence. Association mapping showed five SNPs associated with fungal virulence on chromosome 1, 2, 3, 4 and 7. The SNP on chromosome 1 was associated with virulence against RD6-Pi7 and IRBL7-M which might be linked to the previously reported AvrPi7.     

Overexpression of BAS1 in rice blast fungus can promote blast fungus growth,sporulation and virulence in planta

Background: BAS1 is biotrophy-associated secreted protein of rice blast strain (Magnaporthe oryzae). In order to study the effect of BAS1 on virulence of rice blast strain, we characterized function of BAS1 using a purified prokaryotic expression product of BAS1 and its overexpression strain. Results: Our results showed in vitro the purified expression product caused rapid callose deposition and ROS production in rice leaves and calli, indicated it triggered transient basal defense. When the purified expression product of BAS1 was sprayed onto rice leaves, and 24 h later the leaves were inoculated with blast strain, the results showed the size and number of lesions, on purified BAS1 product-pretreated leaves of the Lijiangxintuanheigu (LTH) challenged with blast strain, was higher than those in BAS1-untreated leaves directly challenged with the same strain, which suggested the defense response trigged by BAS1 can be overcome by other effectors of the fungus. More severe symptoms, higher sporulation, higher relative fungal growth and more lower expression level of defense-related genes appeared in LTH leaves challenged with overexpression strain 35S:BAS1/Mo-2 than those in LTH inoculated with wild-type strain. Conclusions: These data suggest both in vitro pretreatment with BAS1 prokaryotic expression products and overexpression in blast strains can increase virulence of blast fungus.     

The Sch9 Kinase Regulates Conidium Size,Stress Responses,and Pathogenesis in Fusarium graminearum

Fusarium head blight caused by Fusarium graminearum is an important disease of wheat and barley worldwide. In a previous study on functional characterization of the F. graminearum kinome, one protein kinase gene important for virulence is orthologous to SCH9 that is functionally related to the cAMP-PKA and TOR pathways in the budding yeast. In this study, we further characterized the functions of FgSCH9 in F. graminearum and its ortholog in Magnaporthe oryzae. The ΔFgsch9 mutant was slightly reduced in growth rate but significantly reduced in conidiation, DON production, and virulence on wheat heads and corn silks. It had increased tolerance to elevated temperatures but became hypersensitive to oxidative, hyperosmotic, cell wall, and membrane stresses. The ΔFgsch9 deletion also had conidium morphology defects and produced smaller conidia. These results suggest that FgSCH9 is important for stress responses, DON production, conidiogenesis, and pathogenesis in F. graminearum. In the rice blast fungus Magnaporthe oryzae, the ΔMosch9 mutant also was defective in conidiogenesis and pathogenesis. Interestingly, it also produced smaller conidia and appressoria. Taken together, our data indicate that the SCH9 kinase gene may have a conserved role in regulating conidium size and plant infection in phytopathogenic ascomycetes.     

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.     

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.     

The biotrophy-associated secreted protein 4 (BAS4) participates in the transition of Magnaporthe oryzae from the biotrophic to the necrotrophic phase

The physiological and metabolic processes of host plants are manipulated and remodeled by phytopathogenic fungi during infection, revealed obvious signs of biotrophy of the hemibiotrophic pathogen. As we known that effector proteins play key roles in interaction of hemibiotrophic fungi and their host plants. BAS4 (biotrophy-associated secreted protein 4) is an EIHM (extrainvasive hyphal membrane) matrix protein that was highly expressed in infectious hyphae. In order to study whether BAS4 is involved in the transition of rice blast fungus from biotrophic to necrotrophic phase, The susceptible rice cultivar Lijiangxintuanheigu (LTH) that were pre-treated with prokaryotic expression product of BAS4 and then followed with inoculation of the blast strain, more serious blast disease symptom, more biomass such as sporulation and fungal relative growth, and lower expression level of pathogenicity-related genes appeared in lesion of the rice leaves than those of the PBS-pretreated-leaves followed with inoculation of the same blast strain, which demonstrating that BAS4 invitro changed rice defense system to facilitate infection of rice blast strain. And the susceptible rice cultivar (LTH) were inoculated withBAS4-overexpressed blast strain, we also found more serious blast disease symptom and more biomass also appeared in lesion of leaves inoculated with BAS4-overexpressed strain than those of leaves inoculated with the wild-type strain, and expression level of pathogenicity-related genes appeared lower in biotrophic phase and higher in necrotrophic phase of infection, indicating BAS4 maybe in vivo regulate defense system of rice to facilitate transition of biotrophic to necrotrophic phase. Our data demonstrates that BAS4 in vitro and in vivo participates in transition from the biotrophic to the necrotrophic phase of Magnaporthe oryzae.