The OsBSK1-2-MAPK module regulates blast resistance in rice

Receptor-like cytoplasmic kinase OsBSK1-2 was reported to play an important role in regulation of response to rice blast,but the signaling pathway remained unknown.In this study,we identified OsMAPKKK18 and previously uncharacterized MAPKKKs OsMAPKKK16 and OsMAPKKK19 that interact with OsBSK1-2.Expression of all three MAPKKKs was induced by Magnaporthe oryzae infection,and all three induced cell death when transiently expressed in Nicotiana benthamiana leaves.Knockout of OsMAPKKK16 and OsMAPKKK18 compromised blast resistance and overexpression of OsMAPKKK19 increased blast resistance,indicating that all three MAPKKKs are involved in regulation of rice blast response.Furthermore,both OsMAPKKK16 and OsMAPKKK19 interacted with and phosphorylated OsMKK4 and OsMKK5,and chitin-induced MAPK activation was suppressed in osmapkkk16 and osbsk1-2 mutants.OsMAPKKK18 was earlier reported to interact with and phosphorylate OsMKK4 and affect chitin-induced MAPK activation,suggesting that OsBSK1-2 is involved in regulation of immunity through multiple MAPK signaling pathways.Unlike BSK1 in Arabidopsis,OsBSK1-2 was not involved in response to avirulent M.oryzae strains.Taken together,our results revealed important roles of OsMAPKKK16/18/19 and a OsBSK1-2-OsMAPKKK16/18/19-OsMKK4/5 module in regulating response to rice blast.

Physiological Macro-lesions Enhanced Resistance to Blast (Magnaporthe grisea) in Rice Near-isogenic Lines

Roll-leaf-1 （rl-1） and spot-leaf-1 （spl-1） were two near-isogenic lines, which were obtained after 3 to 4 backcrosses with early season indica rice Zhefu 802 as recurrent parent. Henna macro-lesions, referred as physiological or morphological markers, began to appear on leaves at 4.5- to 6.0-leaf stage. The rice seedlings were inoculated at 3.5-, 5.0- and 7.0-leaf stages with high pathogenic races Zhong A1 and Zhong B1 of Magnaporthe grisea, respectively. The resistance of rl-1, spl-1 and Zhefu 802 against blast was significantly different. The seedlings of Zhefu 802 at 3.5- to 7.0-leaf stage were susceptible to races Zhong A1 and Zhong B1 of M. grisea, whereas those of rl-1 and spl-1 at 3.5-, 5.0- and 7.0-leaf stages were susceptible, moderately resistant and resistant, respectively. These results suggested that the enhanced resistance of rl-1 and spl-1 related to the appearance of their morphological marker lesions. The experiment provided a basis for studying lesion mimic and hypersensitive response in association with disease resistance.

Resistance Evaluation of Some Chinese Leading Rice Maintainer, Restorer lines and Their Hybrids to Magnaporthe grisea

Six isolates of Magnaporthe grisea were selected to inoculate on 10 Chinese leading maintainer lines (B-lines), 14 restorer lines (R-lines) and their F1 hybrid plants. In the tested rice materials, R-lines were proved to be more resistant to blast than B-lines. The resistance frequency of about 25% F1 hybrid plants was less than their parents. In addition, 26 isolates of M. grisea collected from different rice growing areas of China were inoculated on 13 new improved hybrid rice combinations. The resistance frequencies of 5 improved hybrids were better than those of the controls and leading varieties in rice production of China.

Expression of a Magnaporthe grisea Elicitor and Its Biological Function in Activating Resistance in Rice

The expression of a protein elicitor from Magnaporthe griesea and its biological function in activating resistance in rice （Oryza safiva L） were reported. The gene of elicitor was expressed in Escherichia colicells and produced a His6-fusion protein with 42 kD apparent molecular weight on SDS-PAGE. The purified protein could induce the resistance to blast disease, with the control efficiency of 46.47% and 36.41% at the 14^th day and the 21^st day after blast inoculation, respectively. After treatment with the expressed protein, the phenylalanine ammonia-lyase （PAL） and peroxidase （POD） activities were promoted in rice plants, meanwhile, the transcription levels of STKM, FAD, PBZ1 and PR1 genes were increased in rice plants. Moreover, after comparing the profile of total rice leaf proteins on two-dimensional electrophoresis gel, about 14 proteins were found to be increased in expression level after the expressed protein treatment. All the results indicated that the expressed protein could act as an elicitor to trigger the resistance in rice.

Resistance Evaluation of Some Hybrid Rice, Conventional Early Indica and Late Japonica Rice to Magnaporthe grisea

Thirty isolates of Magnaporthe grisea collected from 18 provinces/cities representing 21 pathotypes and 9 different lineages were inoculated to rice varieties with known resistance genes and some hybrid rices, conventional early indica and late japonica varieties cultivated recently in China. Virulence spectrum of the 30 isolates was very different, showing that they recognize numerous different resistance genes. Varieties also revealed very different resistance patterns showing that they carry different resistance genes or combinations of resistance genes. On the basis of comparisons with international differential varieties with known resistance genes, resistance genes in certain Chinese varieties could be speculated. The results indicated that some of them were resistant to most of the isolates tested and that they could be of interest as resistance sources for hybrid parents or to be planted in the field directly.

<i>In Vitro</i>Efficacy of Botanicals against Rice Blast Pathogen <i>Magnaporthe oryzae oryzae</i>

Rice is the most staple cereal crop of Bangladesh and rice blast caused by Magnaporthe oryzae oryzae (MoO) has become a major factor limiting rice yield in Bangladesh and throughout the world. Eight botanicals extracted both in water and ethanol namely Kalijira (Nigella sativa), Turmeric (Curcuma longa), Ginger (Zingiber officinalis), Garlic (Allium sativum), Onion (Allium cepa), Neem (Azadirachta indica), Allamanda (Allamanda cathartica) and Aloevera (Aloe vera) were tested against MoO in vitro in the Mycology Laboratory, Department of Plant Pathology, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh. All the botanicals significantly reduced radial growth of the tested pathogen. Maximum mycelia growth inhibition of MoO was achieved with water extract of turmeric (1:1 w/v) and ethanol extracts of neem (1:4 w/v) with 86.57% and 92.62% mycelia growth inhibition at 14 DAI, respectively.

Comparative Analysis of the Genomes of Three Field Isolates of the Rice Blast Fungus <i>Magnaporthe oryzae</i>from Southern China

Rice blast caused by Magnaporthe oryzae (M. oryzae) is one of the most destructive diseases, which causes significant rice yield losses and affects global food security. To better understand genetic variations among different isolates of M. oryzae in the nature field, we re-sequenced and analyzed the genomes of three field isolates, QJ08-2006, QJ10-10, and QJ10-3001, which showed distinct pathogenicity on Xin-Yin-Zhan, an elite variety in South China. Genome annotation indicated that these three isolates assemblies have similar genome sizes with 38.4 Mb, 38.3 Mb, and 38.4 Mb, respectively. The QJ08-2006 assembly has 2082 contigs with an N50 of 127.4 kb, the QJ10-10 assembly has 2239 contigs with an N50 of 105.13 kb, the QJ10-3001 assembly has 2025 contigs with an N50 of 133.16 kb. A total of 10,432 genes including 1408 putative secreted protein genes were identified from the annotated isolate QJ08-2006 genome, 10,418 genes including 1410 putative secreted protein genes were identified in QJ10-10, and 10,401 genes including 1420 putative secreted protein genes were identified in QJ10-3001. There are as many as 11,076 identical genes in these three isolates and contained only a few unique genes among three isolates, of which 277 unique genes in QJ08-2006 and 264 unique genes in QJ10-10, and 213 unique genes in QJ10-3001. Most of the predicted secreted protein genes had been identified, and the three re-sequenced strains contained 371, 369, and 387 small Indel, respectively. Avr genes were analyzed in several sequenced Magnaporthe strains, the results revealed that Avr-Pi9 and Avr-Piz-t were present in all the sequenced isolates. The isolates QJ08-2006 contained AvrPib, QJ10-10, and QJ10-3001 had an insertion of a Pot3 element in the promoter of the AvrPib gene. Our results showed that, the rapid dominancy of virulence mutant isolates via clonal propagation displayed in the field after the release of the elite variety Xin-Yin-Zhan.

Isolating cDNA clones from rice induced by Magnaporthe grisea using PCR based differential screening method

The differential hybridization technique hasbeen widely used to identify genes that are dif-ferentially expressed.However,this approachhas several drawbacks.First,the screeningprocedures are rather labor-intensive and time-consuming.Second,the amount of phageDNAs transferred onto the two filters may notbe equivalent,which leads to an inaccurate se-lection of a positive clone.Third,isolation ofphage DNA is slow and cumbersome.Here,aPCR based differential screening method that

稻瘟病菌(Magnaporthe grisea)诱导的水稻早期反应基因ER1的5’端cDNA片段克隆及序列分析

The 5’ end cDNA fragment(ER1’) of early responsive gene(ER1) of rice(Oryza sativa L. subsp. japonica No.4) induced by blast fungus M. grisea has been isolated and sequenced (606 bp) by 5’RACE technique(Fig. 2). DNA sequence analysis showed that ER1’ (606 bp) can encode 134 amino acids and there are 309 bp nucleotides located at the 3’ end of the untranslated region of mRNA of ER1 gene(Fig. 3). The 89 bp overlapping fragment was found between the 5’ end of ER1 cDNA fragment and the 3’ end of ER1’(Fig. 3). The partial identities of amino acid sequence deduced from ER1’ with an unknown protein(gene accession number AC002409) of Arabidopsis thaliana was 82%(Fig. 4).

Identification and fine mapping of two blast resistance genes in rice cultivar 93-11

Rice blast, caused by Magnaporthe oryzae, is a major disease of rice almost worldwide. The Chinese indica cultivar 93-11 is resistant to numerous isolates of the blast fungus in China, and can be used as broad-spectrum resistance resource, particularly in japonica rice breeding programs. In this study, we identified and mapped two blast resistance genes, Pi60(t) and Pi61(t), in cv. 93-11 using F2 and F3 populations derived from a cross between the susceptible cv. Lijiangxintuanheigu(LTH) and resistant cv. 93-11 and inoculated with M. oryzae isolates from different geographic origins. Pi60(t) was delimited to a 274 kb region on the short arm of chromosome 11, flanked by InDel markers K1-4 and E12 and cosegregated with InDel markers B1 and Y10. Pi61(t) was mapped to a 200 kb region on the short arm(near the centromere) of chromosome 12, flanked by InDel markers M2 and S29 and cosegregating with InDel marker M9. In the 274 kb region of Pi60(t), 93-11 contains six NBS-LRR genes including the two Pia/ PiCO39 alleles(BGIOSGA034263 and BGIOSGA035032) which are quite close to the two Pia/ PiCO39 alleles(SasRGA4 and SasRGA5) in Sasanishiki and CO39, with only nine amino acids differing in the protein sequences of BGIOSGA035032 and SasRGA5. In the 200 kb region of Pi61(t), 93-11 contains four NBS-LRR genes, all of which show high identities in protein sequence with their corresponding NBS-LRR alleles in susceptible cv. Nipponbare. Comparison of the response spectra and physical positions between the target genes and other R genes in the same chromosome regions indicated that Pi60(t) could be Pia/PiCO39 or its allele, whereas Pi61(t) appears to be different from Pita, Pita-2, Pi19(t), Pi39(t) and Pi42(t) in the same R gene cluster. DNA markers tightly linked to Pi60(t) and Pi61(t) will enable marker-assisted breeding and map-based cloning.

Analysis of the Abnormal Segregation of Pathogenicity in Magnaporthe grisea by Using a Genetic Cross of Oryza and Eleusine Isolates

A genetic cross between Oryza isolate Y93-164a-1 and Eleusine isolate SA98-4 was established, and the pathogenicity of 151 F1 progeny isolates was investigated on both host plants rice and finger millet. Results showed that the segregation of pathogenicity in this genetic cross was abnormal, i.e., most of the progeny isolates were nonpathogenic on both host plants. However, no abnormal segregation was observed when middle repetitive sequence MGR586 and 31 single-copy RFLP markers from all of the chromosomes were genetically analyzed. At the same time, comparison of the chromosomal organization among two pairs of parental isolates did not find any genomic abnormity. These results suggested that the ＂abnormal＂ inheritance of pathogenicity in this cross was most likely due to the reassortment of numerous host species specificity genes but not the biased segregation of the host species specificity genes. The host species specificities in M. grisea were likely to be multigenically controlled, at least in the genetic cross involving rice pathogen and the grasses pathogen other than rice.

Host Active Defense Responses Occur within 24 Hours after Pathogen Inoculation in the Rice Blast System

Phenotypical, cytological and molecular responses of rice to the fungus Magnaporthe grisea were studied using rice cultivars and lesion mimic plants. The cultivar Katy was susceptible to several virulent M. grisea isolates, and a Sekiguchi like-lesion mimic mutant of Katy （LmmKaty） showed enhanced resistance to these isolates. Lesion mimic phenotype of LmmKaty was rapidly induced by virulent M. grisea isolates or by avirulent ones only at high levels of inoculum. Autofluorescence （a sign of an active defense response） was visible under ultraviolet light 24 h after localized inoculation in the incompatible interaction, whereas, not evident in the compatible interaction. Autofluorescence was also observed in LmmKaty 20 h after pathogen inoculation, indicating that rapid cell death is a mechanism of LmmKaty to restrict pathogen invasion. Rapid accumulations of defense related （DR） gene transcripts, phenylalanine ammonia lyase and β-glucanase, were observed beginning at 6 h and were obvious at 16 h and 24 h after inoculation in an incompatible interaction. Rapid transcript accumulations of PR-1 and chitinase had occurred by 24 h after inoculation in an incompatible interaction. Accumulations of these transcripts were delayed in the compatible interaction. These results indicate that host active defense responses occur 24 h after pathogen inoculation and that LmmKaty exhibits enhanced resistance to M. grisea. It is suggested that the autofluorescence and expression of the DR genes after heavy inoculation are important cytological and molecular markers respectively for early determination of the host response to M. grisea in the rice blast system.

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.

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.

Pathotype Structure of Magnaporthe grisea in the Fields of Chongyang and Yuan'an in Hubei Province,China

[Objective] This study aimed to explore the pathotype structure of Magnaporthe grisea in Chongyang and Yuan'an in Hubei Province,China.[Method] From the rice-growing fields of Chongyang and Yuan'an in Hubei Province where rice blast occurs frequently,60 isolates which were pathotyped against two sets of host differentials:Chinese host differentials and CO39 NILs,were obtained.Then,20 pathotypes with the six indica host differentials(CO39 NILs) were observed,while 13 pathotypes in four race groups were observed out of the same single spore isolates with Chinese host differentials which consists of three indica cultivars and four japonica cultivars.The diversity of the pathotypes of M.grisea populations tested by CO39 NILs was 2.54 and the pathotype 137.1 occurred at predominantly high frequency(21.67%).The diversity of physiological races of M.grisea populations tested by Chinese host differentials was 1.22 and the race group ZA occurred at predominantly high frequency(73.33%).The diversity of physiological races of M.grisea in Chongyang and Yuan'an were also calculated.Overall,the diversity of pathotypes of M.grisea in Yuan'an was higher than that in Chongyang with the two sets of the host differentials.[Conclusion] This study provided current information on the pathotype spectrum of M.grisea populations in the rice fields of Hubei Province to allow the formulation of viable strategies for blast resistance breeding programs in Hubei Province.

Functional genomics in the rice blast fungus to unravel the fungal pathogenicity

A rapidly growing number of successful genome sequencing projects in plant pathogenic fungi greatly increase the demands for tools and methodologies to study fungal pathogenicity at genomic scale. Magnaporthe oryzae is an economically important plant pathogenic fungus whose genome is fully sequenced. Recently we have reported the development and application of functional genomics platform technologies in M. oryzae. This model approach would have many practical ramifications in design and implementation of upcoming functional genomics studies of filamentous fungi aimed at understanding fungal pathogenicity.

Genome-Wide Analysis of von Willebrand Factor A Gene Family in Rice for Its Role in Imparting Biotic Stress Resistance with Emphasis on Rice Blast Disease

von Willebrand factor A(vWA)genes are well characterized in humans except for few BONZAI genes,but the vWA genes are least explored in plants.Considering the novelty and vital role of vWA genes,this study aimed at characterization of vWA superfamily in rice.Rice genome was found to have 40 vWA genes distributed across all the 12 chromosomes,and 20 of the 40 vWA genes were unique while the remaining shared large fragment similarities with each other,indicating gene duplication.In addition to vWA domain,vWA proteins possess other different motifs or domains,such as ubiquitin interacting motif in protein degradation pathway,and RING finger in protein-protein interaction.Expression analysis of vWA genes in available expression data suggested that they probably function in biotic and abiotic stress responses including hormonal response and signaling.The frequency of transposon elements in the entire 3K rice germplasm was negligible except for 9 vWA genes,indicating the importance of these genes in rice.Structural and functional diversities showed that the vWA genes in a blast-resistant rice variety Tetep had huge variations compared to blast-susceptible rice varieties HP2216 and Nipponbare.qRT-PCR analysis of vWA genes in Magnaporthe oryzae infected rice tissues indicated OsvWA9,OsvWA36,OsvWA37 and OsvWA18 as the optimal candidate genes for disease resistance.This is the first attempt to characterize vWA gene family in plant species.

Inhibitory Activity of <i>Burkholderia</i>sp. Isolated from Soil in Gotsu City, Shimane, against <i>Magnaporthe oryzae</i>

An isolate GT4028 was obtained from soil samples collected from a field in Gotsu city (Kawahira), Shimane. The use of a culture suspension and culture filtrate of this isolate significantly suppressed the spore germination in Magnaporthe oryzae. The inhibitory activity of the culture filtrate was heat-stable. The formation of rice blast lesions by M. oryzae was significantly suppressed in the presence of the culture suspension of isolate GT4028. Furthermore, mycelial growth of some plant pathogenic fungi was inhibited by the isolate in a dual culture assay. Sequence analysis of 16S rDNA region of the isolate indicated that it shared similarities with species of the genus Burkholderia. Also, isolate GT4028 could be grown even in the presence of fungicides (Blastin, Kasugamycin, and Amistar) that act against M. oryzae. These results suggest that isolate GT4028 might be a potential control agent for plant protection against diseases, such as rice blast disease.

CRISPR/Cas9-Targeted Knockout of Rice Susceptibility Genes OsDjA2 and OsERF104 Reveals Alternative Sources of Resistance to Pyricularia oryzae

Rice genes OsDjA2 and OsERF104,encoding a chaperone protein and an APETELA2/ethylene-responsive factor,respectively,are strongly induced in a compatible interaction with blast fungus,and also have function in plant susceptibility validated through gene silencing.Here,we reported the CRISPR/Cas9 knockout of OsDjA2 and OsERF104 genes resulting in considerable improvement of blast resistance.A total of 15 OsDjA2(62.5%)and 17 OsERF104(70.8%)T_(0)transformed lines were identified from 24 regenerated plants for each target and used in downstream experiments.Phenotyping of homozygous T1 mutant lines revealed not only a significant decrease in the number of blast lesions but also a reduction in the percentage of diseased leaf area,compared with the infected control plants.Our results supported CRISPR/Cas9-mediated target mutation in rice susceptibility genes as a potential and alternative breeding strategy for building resistance to blast disease.