The DNA damage repair complex MoMMS21-MoSMC5 is required for infection-related development and pathogenicity of Magnaporthe oryzae

The conserved DNA damage repair complex,MMS21-SMC5/6(Methyl methane sulfonate 21-Structural maintenance of chromosomes 5/6),has been extensively studied in yeast,animals,and plants.However,its role in phytopathogenic fungi,particularly in the highly destructive rice blast fungus Magnaporthe oryzae,remains unknown.In this study,we functionally characterized the homologues of this complex,MoMMS21 and MoSMC5,in M.oryzae.We first demonstrated the importance of DNA damage repair in M.oryzae by showing that the DNA damage inducer phleomycin inhibited vegetative growth,infection-related development and pathogenicity in this fungus.Additionally,we discovered that MoMMS21 and MoSMC5 interacted in the nuclei,suggesting that they also function as a complex in M.oryzae.Gene deletion experiments revealed that both MoMMS21 and MoSMC5 are required for infection-related development and pathogenicity in M.oryzae,while only MoMMS21 deletion affected growth and sensitivity to phleomycin,indicating its specific involvement in DNA damage repair.Overall,our results provide insights into the roles of MoMMS21 and MoSMC5 in M.oryzae,highlighting their functions beyond DNA damage repair.

The Magnaporthe oryzae effector Avr-PikD suppresses rice immunity by inhibiting an LSD1-like transcriptional activator

Avirulence effectors(Avrs),encoded by plant pathogens,can be recognized by plants harboring the corresponding resistance proteins,thereby initiating effector-triggered immunity(ETI).In susceptible plants,however,Avrs can function as effectors,facilitating infection via effector-triggered susceptibility(ETS).Mechanisms of Avr-mediated ETS remain largely unexplored.Here we report that the Magnaporthe oryzae effector Avr-PikD enters rice cells via the canonical cytoplasmic secretion pathway and suppresses rice basal defense.Avr-PikD interacts with an LSD1-like transcriptional activator AKIP30 of rice,and AKIP30 is also a positive regulator of rice immunity,whereas Avr-PikD impedes its nuclear localization and suppresses its transcriptional activity.In summary,M.oryzae delivers Avr-PikD into rice cells to facilitate ETS by inhibiting AKIP30-mediated transcriptional regulation of immune response against M.oryzae.

Ecofriendly Management of Wheat Panicle Blast Caused by Magnaporthe oryzae triticum

In this study, three wheat varieties were tested to determine seed germination and the incidence of Magnaporthe oryzae triticum (MoT). Among these varieties, BARI Gom 24 (Prodip) wheat seed exhibited the highest seed germination rate (93%) but also had the highest incidence (30%) of MoT. To manage blast disease in an ecofriendly manner, seven treatments were employed: T1 = Control, T2 = Garlic clove extracts, T3 = Aloe vera leaf extracts, T4 = Black cumin seed extracts, T5 = Neem leaf extracts, T6 = Nativo 75 WG, and T7 = Provax 200 WP. The experiment was conducted using a Randomized Complete Block Design (RCBD) layout with three replications using Prodip wheat variety that exhibited highest MoT infection severity based on laboratory analysis among collected varieties. Data were collected on blast disease incidence (%), disease severity, and various growth and yield parameters of wheat. The experiment’s results indicated that among all the treatments, T7 (Seed treatment with Provax 200 WP) and T5 (Foliar spraying with Neem leaf extract) performed better in controlling blast disease in wheat. The lowest blast disease incidence (%) was observed with T7 (Provax 200 WP), with values of 7.86, 9.86, and 10.19 recorded during the milking stage, soft dough stage, and hard dough stage of wheat, respectively. T5 (Neem leaf extract) also demonstrated a statistically equivalent reduction in blast disease incidence (%). In terms of disease severity, T7 (Seed treatment with Provax 200 WP) showed the lowest values of 1.03, 1.23, and 1.63 during the milking stage, soft dough stage, and hard dough stage of wheat, respectively. Foliar spraying with neem leaf extract also exhibited similar result as of Provax 200 WP regarding panicle blast severity. As a result of these findings, it can be concluded that T5 (Neem leaf extract) is recommended as an ecofriendly management approach for blast disease in wheat.

Synthetical Evaluation on the Qualitative and Quantitative Resistance of Rice Germplasms to Magnaporthe oryzae

[Objectives]This study was conducted to screen out rice resources resistant to rice blast(Magnaporthe oryzae).[Methods]The qualitative and quantitative resistance of 1659 rice resources from 45 countries and regions to rice blast were evaluated by disease nursery in upland condition and the test of the spectrum to rice blast isolates.[Results]There were 292 entries which accounted for 17.6%showed high blast resistance(0 and 1 disease scale),68 entries(counted for 4.1%)showed resistance to blast(3 disease scale);and the number of the entries showed intermediate resistance,intermediate susceptible and susceptible were 208(with the corresponding percentage of 12.5%),471(28.4%),620(37.4%)respectively.Among the tested entries,27 entries including BG1222,BL122,BTX,IR37704-131-2-3-2,and LEBONNET had showed broad-spectrum blast resistance with the resistance frequency of higher than 90%,Quantitative resistance evaluation was conducted on some key resources,and 14 entries,of which are BR27,DRAGO,IR100,QINLIUAI、SERIBU GANTANG,YUEXIANGZHAN and so on,showed good quantitative resistances,and 8 entries had higher quantitative resistances than IR36.[Conclusions]This study provides important blast resistance resources for the local rice breeding program and has a significant value for the discovery of new blast resistance genes and its application in the blast resistance breeding.

Population Genetic Structure of Magnaporthe oryzae in Rice Blast Epidemic Areas in Hubei Province

Single-spore isolates were obtained from rice-growing fields of Yuan＇an in Hubei Province where rice blast seriously occurs in some years. DNA fingerprints were divided into 112 haplotypes and 14 lineages at 73% genetic similarity level. Among the lineages, no dominant lineages were found. The population genetic structures of Magnaporthe oryzae were not distinctly different in different years. The analysis also showed that there wasn＇t obvious simple relationship between patho- types and fingerprint groups.

Fluorescent co-localization of PTS1 and PTS2 and its application in analysis of the gene function and the peroxisomal dynamic in Magnaporthe oryzae

The peroxisomal matrix proteins involved in many important biological metabolism pathways in eukaryotic cells are encoded by nucleal genes, synthesized in the cytoplasm and then transported into the organelles. Targeting and import of these proteins depend on their two peroxisomal targeting signals （PTS 1 and PTS2） in sequence as we have known so far. The vectors of the fluorescent fusions with PTS, i.e., green fluorescence protein （GFP）-PTS1, GFP-PTS2 and red fluorescence protein （RFP）-PTS1, were constructed and introduced into Magnaporthe oryzae Guy ll cells. Transformants containing these fusions emitted fluorescence in a punctate pattern, and the locations of the red and green fluorescence overlapped exactly in RFP-PTS 1 and GFP-PTS2 co-transformed strains. These data indicated that both PTS1 and PTS2 fusions were imported into peroxisomes. A probable higher efficiency of PTS1 machinery was revealed by comparing the fluorescence backgrotmds in GFP-PTS1 and GFP-PTS2 transformants. By introducing both RFP-PTS1 and GFP-PTS2 into Amgpex6 mutants, the involvement of MGPEX6 gene in both PTS1 and PTS2 pathways was proved. In addition, using these transformants, the inducement ofperoxisomes and the dynamic of peroxisomal number during the pre-penetration processes were investigated as well. In summary, by the localization and co-localization of PTS1 and PTS2, we provided a useful tool to evaluate the biological roles of the peroxisomes and the related genes.

Rapid Detection of Wheat Blast Pathogen Magnaporthe oryzae Triticum Pathotype Using Genome-Specific Primers and Cas12a-mediated Technology

Wheat blast,caused by the fungus Magnaporthe oryzae Triticum(MoT)pathotype,is a devastating disease persistent in South America and Bangladesh.Since MoT generally fails to cause visual symptoms in wheat until the heading stage when the infection would have advanced,disease control by fungicide application solely based on the detection of visual symptoms is ineffective.To develop an accurate and sensitive method to detect MoT at the seedling and vegetative stages for disease control,we sequenced the genomes of two MoT isolates from Brazil and identified two DNA fragments,MoT-6098 and MoT-6099,that are present in the MoT genome but not in the genome of the rice-infecting Magnaporthe oryzae Oryzae(MoO)pathotype.Using polymerase chain reaction(PCR),we confirmed the specificity of the two markers in 53 MoT and MoO isolates from South America and Bangladesh.To test the efficiency of the two markers,we first established a loop-mediated isothermal amplification(LAMP)method to detect MoT at isothermal conditions,without the use of a PCR machine.Following this,we used the Cas12a protein and guide RNAs(gRNAs)to target the MoT-6098 and MoT-6099 sequences.The activated Cas12a showed indiscriminate single-stranded deoxyribonuclease(ssDNase)activity.We then combined targetdependent Cas12a ssDNase activation with recombinase polymerase amplification(RPA)and nucleic acid lateral flow immunoassay(NALFIA)to develop a method that accurately,sensitively,and cost-effectively detects MoT-specific DNA sequences in infected wheat plants.This novel technique can be easily adapted for the rapid detection of wheat blast and other important plant diseases in the field.

Investigation of the biological roles of autophagy in appressorium morphogenesis in Magnaporthe oryzae

Magnaporthe oryzae has been used as a primary model organism for investigating fungus-plant interaction. Many researches focused on molecular mechanisms of appressorium formation to restrain this fungal pathogen. Autophagy is a very high conserved process in eukaryotic cells. Recently, autophagy has been considered as a key process in development and differentia-tion in M. oryzae. In this report, we present and discuss the current state of our knowledge on gene expression in appressorium formation and the progress in autophagy of rice blast fungi.

A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1 × 106 ml^-1, the percentages of conidium germination and appressorium formation were （97.98±0.67）% and （97.88±0.45）%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 lag total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA （complementary DNA） library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.

Magnaporthe oryzae MTP1 gene encodes a type Ⅲ transmembrane protein involved in conidiation and conidial germination

In this study the MTP1 gene, encoding a type III integral transmembrane protein, was isolated from the rice blast fungus Magnaporthe oryzae. The Mtp 1 protein is 520 amino acids long and is comparable to the Ytp 1 protein of Saccharomyces cerevisiae with 46% sequence similarity. Prediction programs and MTP1-GFP （green fluorescent protein） fusion expression results indicate that Mtp 1 is a protein located at several membranes in the cytoplasm. The functions of the MTP1 gene in the growth and development of the fungus were studied using an MTP1 gene knockout mutant. The MTP1 gene was primarily expressed at the hyphal and conidial stages and is necessary for conidiation and conidial germination, but is not required for pathogenicity. The Amtpl mutant grew more efficiently than the wild type strain on non-fermentable carbon sources, implying that the MTP1 gene has a unique role in respiratory growth and carbon source use.

Evolutionary analysis of plant jacalin-related lectins(JRLs)family and expression of rice JRLs in response toMagnaporthe oryzae

Jacalin-related lectins （JRLs） are widely distributed carbohydrate-binding proteins in the plant kingdom, which play key roles in development and pathogen defense. In this study, we profiled evolutionary trajectory of JRLs family in 30 plant species and identified domain diversification and recombination leading to different responsive patterns of JRLs in rice during defense against rice blast. All of 30 plant species analyzed in our study have two types of JRLs by containing either a single jacalin or repeated jacalin domains, while chimeric jacalins exist in more than half of the species, especially in the Poaceae family. Moreover, Poaceae species have evolved two types of unique chimeric JRLs by fusing the jacalin domain（s） with dirigent or NB_ARC domain, some of which positively regulate plant immunity. Seven Poaceae-specific JRLs are found in the rice genome. We further found expression of rice JRLs, including four Poaceae-specific JRLs, are induced by Magnaporthe oryzae infections at either early or late infection stages. Overall, the results present the evolutionary trajectory of JRLs in plant and highlight essential roles of Poaceae specific JRLs against pathogen attacks in rice.

Changes in the epigenome and transcriptome of rice in response to Magnaporthe oryzae infection

DNA methylation participates in regulating the expression of coding and non-coding regions in plants. To investigate the association between DNA methylation and pathogen infection, we used whole-genome bisulfite sequencing to survey temporal DNA methylation changes in rice after infection with the rice blast fungus Magnaporthe oryzae. In contrast to previous findings in Arabidopsis, global DNA methylation levels in rice increased slightly after rice blast infection. We identified over 38,000 differentially methylated regions(DMRs), and hypermethylated DMRs far outnumbered hypomethylated DMRs. Most DMRs were located in transposable element regions. Using transcriptome analysis, we identified 8830 differentially expressed genes(DEGs) after 1, 3, and 5 days of infection. Over one-third of DEGs, most of which were CHH-type DMRs, were associated with DMRs. Functional analysis of the CHH DMR-DEGs indicated their involvement in many important biological processes, including cell communication and response to external stimulus. The transcription of many NBS-LRR family genes was affected by changes in DNA methylation, suggesting that DNA methylation plays essential roles in the response of rice to M. oryzae infection. More broadly, the DNA methylation analysis presented here sheds light on epigenomic involvement in plant defense against fungal pathogens.

Comparative analysis of the genome of the field isolate V86010 of the rice blast fungus Magnaporthe oryzae from Philippines

Genome dynamics of pathogenic organisms are driven by plant host and pathogenic organism co-evolution, in which patho- gen genomes areused to overcome stresses imposed by hosts with various genetic backgrounds through generation of a range of field isolates. This model also applies to the rice host and its fungal pathogen Magnaporthe oryzae. To better understand genetic variation of M. oryzae in nature, the field isolate V86010 from the Philippines was sequenced and ana- lyzed. Genome annotation found that the assembled V86010 genome was composed of 1 931 scaffolds with a combined length of 38.9 Mb. The average GC ratio is 51.3% and repetitive elements constitute 5.1% of the genome. A total of 11 857 genes including 616 effector protein genes were predicted using a combined analysis pipeline. All predicted genes and effector protein genes of isolate V86010 distribute on the eight chromosomes when aligned with the assembled genome of isolate 70-15. Effector protein genes are located disproportionately at several chromosomal ends. The Pot2 elements are abundant in V86010. Seven V86010-specific effector proteins were found to suppress programmed cell death induced by BAX in tobacco leaves using an Agrobacterium-mediated transient assay. Our results may provide useful information for further study of the molecular and genomic dynamics in the evolution of M. oryzae and rice host interactions, and for characterizing novel effectors and AVR genes in the rice blast pathogen.

Integrated pest management programme for cereal blast fungus Magnaporthe oryzae

Magnaporthe oryzae,the causal agent of blast diseases,is a destructive filamentous fungus that infects many plants including most economically important food crops,rice,wheat,pearl millet and finger millet.Magnaporthe oryzae has numerous pathotypes because of its high host-specificity in the field.The Oryza pathotype(MoO)of M.oryzae is the most devastating pathogen of rice,causing 10–30%yield loss in the world.On the other hand,the Triticum pathotype(MoT)causes blast disease in wheat,which is now a serious threat to wheat production in some South American countries,Bangladesh and Zambia.Because of low fungicide efficacy against the blast diseases and lack of availability of resistant varieties,control of rice and wheat blast diseases is difficult.Therefore,an integrated management programme should be adopted to control these two diseases in the field.Here,we introduced and summarized the classification,geographical distribution,host range,disease symptoms,biology and ecology,economic impact,and integrated pest management(IPM)programme of both rice and wheat blast diseases.

Pathogenicity of Rice Blast Fungus Magnaporthe oryzae on Brachypodium distachyon

Inoculation methods for rice blast fungus Magnaporthe oryzae to Brachypodium distachyon were developed to investigate the infection process and symptom development in comparison with those on rice （Oryza sativa） and barley （Hordeum vulgare）.M.oryzae could infect leaves,sheathes,stems and panicles of B.distachyon and cause blast disease.Spraying conidial suspension on either intact seedlings or leaf segments induced typical symptoms on B.distachyon.During the intact seedling inoculation,the symptom developed on B.distachyon leaves closely resembled that on rice;but the lesions on B.distachyon had better uniformity in shapes and sizes than those on rice or barley.In the leaf segments inoculation,only initial and low-developed lesions could be found on rice,while normal symptoms on B.distachyon and barley.Inoculated with low-virulent mutants of M.oryzae,B.distachyon produced low-level symptoms.The symptom level of each mutant on B.distachyon corresponded well to that on rice.In addition,typical infection processes presented on B.distachyon leaves：forming melanized appressoria,penetrating into host epidermis and then forming hyphae in epidermal cells.According to these results,B.distachyon can be used as a candidate for studying fungus-plant interactions and as a probable source of disease resistance.

Virulence Types of Magnaporthe oryzae to Hybrid Rice in Sichuan,China

A total of 638 isolates of rice blast （Magnaporthe oryzae） were isolated in 2002-2009 from different rice varieties in different regions of Sichuan, China and inoculated onto seven rice varieties （Lijiangxintuanheigu, IR24, Minghui 63, Duohui 1, Chenghui 448, Neihui 99-14 and RHR-1） to differentiate the virulence types of the fungus and trace the changes. The virulence to the seven varieties was respectively scored at 1, 2, 4, 8, 16, 32 and 64. The total scores of individual M. grisea isolates which were the sum of scores infecting differential varieties could, in turn, be used for the nomenclature of the virulence types due to their accordance to the special virulence patterns. The 638 tested isolates were then differentiated into 56 different virulence types. Type 15 virulent to Lijiangxintuanheigu, IR24 and Minghui 63, and Type 127 virulent to all of the seven varieties were the most dominant virulence types respectively with the occurrence frequencies of 15.99% and 15.83%. Type 19 and other seven virulence types were not monitored during 2002-2009. Type 15 was the predominant virulence type in 2002, 2003, 2004 and 2007, whereas Type 127 had been the most dominant virulence type after 2005 except for the year 2007 when the province underwent severe drought. Five hundred and seven out of the 638 tested isolates were virulent to Minghui 63, and 89.58% of the 384 isolates virulent to either Duohui 1, Chenghui 448 or Neihui 99-14 were virulent to Minghui 63, which indicated the impact of the extensive plantation of hybrid rice Minghui 63 as the restorer line on the virulence evolution of M. oryzae in Sichuan. The virulence pattern of the dominant virulence types suggested that the acquiring of virulence to all the major resistant restorer lines was the main routes of the evolution in virulence of M. oryzae to hybrid rice in Sichuan. The virulence frequencies of the 638 tested isolates to IR24, Minghui 63, Duohui 1, Chenghui 448, Neihui 99-14 and RHR-1 were respectively 74.6%, 79.5%, 73.8%, 37.0%, 39.0% and 40.4%. The analysis for the sources of the different virulence type isolates indicated the pathogen on the newly released resistant varieties were stronger than conventional rice varieties which had become susceptible in the field since 1980s.

Identification and Analysis of Physiological Races of Magnaporthe oryzae in Heilongjiang Province

A totatl of 116 isolates of rice blast fungus, Magnaporthe oryzae, were collected from 45 samples in different counties of Heilongjiang Province, and 20 Chinese physiological races belonging to seven groups were identified by using seven standard Chinese rice blast identifying varieties. Results showed that the dominant groups could be ranked as ZA, ZD, and ZB, with the occurrence frequencies of 47.41%, 22.41% and 15.52%, respectively. The race ZA49 was the dominant race with the occurrence frequency of 26.72%. The occurrence frequencies of the races ZD5 and ZD1 were 10.34% and 8.62%, respectively. The rising occurrence frequencies of these three dominant species were the most important reasons that causing Kongyu-131 more sensitive to rice blast. The results of virulence frequency indicated that the race harboring Pi-k, Pi-i, Pi-a gene were more susceptible to rice blast in Heilongjiang Province, and they should not be large-scale cultivated.

Functional Characterization of a NEM1-Like Gene in Magnaporthe oryzae

Magnaporthe oryzae, a filamentous ascomycete fungus, is well known as the causal agent of rice blast. With the technology of suppression subtractive hybridization （SSH）, it was previously found that MGG_06001 （or named MoNEM1）, a gene of M. oryzae homologous to the NEM1 （nuclear envelope morphology protein 1） gene of baker＇s yeast （Saccharomyces cerevisiae）, is differentially expressed between the mature appressium and the conidium and mycelium. This study aimed to characterize the function of MoNEM1 gene by knocking it out using the method of target gene replacement. The AMoneml mutants exhibited reduced mycelial growth and conidiation. However, disruption of MoNEM1 gene does not affect the pathogenicity of M. oryzae on barley and rice.

Isolation of Gene Mutation from a Pathogenicity-Enhanced Mutant of Magnaporthe oryzae

To find new genes involved in fungal pathogenicity, a mutant （B11 ） exhibiting enhanced pathogenicity was isolated from an Agrobacterium-mediated transformed Magnaporthe oryzae mutant library. Southern blotting analysis showed that T-DNA insertion in the B11 genome was a single copy. TAIL-PCR and sequence alignment analyses revealed that a putative gene locus MG01679 was interrupted by the T-DNA fragment. By using the PCR-based method, the DNA and cDNA of the mutant gene MG01679 was cloned and sequenced. The open reading frame of MG01679 includes one intron and two exons, and the coding sequence is 696 bp in length and encodes a 231 amino acid peptide. Protein similarity analysis indicated that the gene belongs to the ThiJ/Pfp I protein family, and the gene was thus designated MgThiJ1. MgThiJ1 showed 57% similarity to FOXG_09029 from Fusarium oxysporum and 54% similarity to FGSG_08979 from F. graminearum in protein sequence. MgThiJ1 gene might act as a negative regulator in vegetative growth and pathogenesis in filamentous fungi, and its specific mechanism needs to be studied further.

Culture Filtrates of Trichoderma Isolate H921 Inhibit Magnaporthe oryzae Spore Germination and Blast Lesion Formation in Rice

Spore germination and appressorium formation of Magnaporthe oryzae spores was completely suppressed by an ethyl acetate extract of the culture filtrate from the H921 isolate (H921-EAE-CF). Production of antifungal substance(s) in the H921-EAE-CF began to increase up to 3 days after isolate H921 incubation. Furthermore, heat treatment (105°C or 121°C) of H921-EAE-CF did not alter its inhibitory effect on M. oryzae spore germination compared to non-heat-treated H921-EAE-CF. Blast lesion formation inhibition by H921-EAE-CF was dose-dependent. Internal transcribed spacer (ITS) region sequence analysis indicated that this isolate shared similarities with species of the genera Trichoderma. This study suggests that H921-EAE-CF contains some antifungal substances that could be promising candidates for control of rice blast disease.