| Rice(Oryza sativa L.)is one of the most important staple foods for more than half of the world population.Rice blast disease caused by the fungus Magnaporthe grisea(M.grisea)leads to the decrease of rice yields destructively.Annual yield losses of rice destroyed by blast are sufficient to feed 60 million people.Additionally,the blast fungus is also harmful to wheat and other small grains and leads to a large reduction in yield.Many strategies,including fungicides,resistant cultivars and agronomical practices have been developed to overcome this disastrous disease.However,development and use of rice new cultivars with broad-spectrum resistance to rice blast is the most effective and economic way to control the blast disease.The resistant ability of new rice varieties to blast fungus will be lost after several year applications,presumably due to the strong selection pressure for the evolution of virulent races.Thus,it is significant to keep mining novel broad-spectrum resistant genes in rice and use these genes to develop new rice varieties with resistant ability to blast fungus.In this study,rice cultivars CO39(Oryza sativa L.indica)and its 5 Near-Isogenic Lines(NILs),including C101 LAC,C101A51,C104 PKT,C101PKT and C105TTP-4L-23(named Pi1,Pi2,Pi3,Pi4 a and Pi4 b,respectively)were employed as materials.Based on the genome re-sequencing and transcription sequencing of the test rice varieties under the infection of blast fungus,the candidate blast-resitant genes in rice were screened through genomic SNP and differentially expressed genes(DEGs)analysis.After that,rice cultivar,Pi4 b,with highly resistant ability to GUY11 was used to construct mutants by CRISPR/Cas9 to identify the function of the candidate blast-resistant genes and elucidate their molecular mechanism.In addition,the mutant d1-Pi4 b of G? was obtained to discover the signal transduction of G? in rice under blast fungus infection.The results were summaried at below.1.OsBRG1 is a novel blast-resistant gene in rice and could simultaneously regulate the development of rice seeds.The candidate gene OsBRG1 was selected from the comparative genomics analysis of the test rice varieties.First,OsBRG1 mutant lines were developed from Pi4 b using CRISPR/Cas9,and these mutants were susceptible to GUY11 7dpi with rank 4(susceptible),while Pi4 b was resistant to GUY11 with rank 1(resistant).The result indicated that OsBRG1 was a new blast-resistant gene in rice.OsBRG1 gene was found to encode a NBS-LRR protein with typical conserved domains NBS and LRR,and it was subcellular localized in the cytoplasm and cell membrane of Nicotiana benthamiana(N.benthamiana)cells and rice protoplasts.The hypersensitive response of OsBRG1 further proved that OsBRG1 was resistant to GUY11.Finally,the molecular mechanism of OsBRG1's resistant ability to GUY11 was analyzed.OsBRG1 was up-regulated under SA,MeJA,ET and ABA treatment,indicating that this gene was involved in the response to biotic and abiotic stress of rice.OsBRG1 was up-regulated in susceptible and resistant rice cultivars under the infection of GUY11,proving that this gene participated in the immune reaction process to biotic stress of rice.Whilst,OsBRG1 was also up-regulated by high temperature stress,showing that that OsBRG1 had ability to response to abiotic stress.The expressing abundance of MAPK6,WRKY45,PR1 a,PR2,PR3 and PR10 in wild type were higher than that in OsBRG1 mutant under the infection of GUY11,respectivelty,manifesting that the blast resistance ability of OsBRG1 was related to the expression of the above genes.Using rice protoplasts system,59 proteins were sepqrated from OsBRG1 protein complex through IP method,among which the direct interaction between Os07g0105900 and OsBRG1 was comfirmed by Y2 H.The result indicated that OsBRG1 could interact with Os07g0105900 to mediate the blast resistant ability in rice.Comparing the seed of OsBRG1 mutant M-1(72bp deletion),M-2 and wildtype,we found that the seed length of M-1 was longer than M-2 and Pi4 b,and M-2 and Pi4 b had no significant difference.The seed width of M-1 and M-2 was smaller than Pi4 b.In addition,the thousand seed weight of M-1 was 25.68 g,which was 10.0%higher than that of wild type.Meanwhile,M-1 had a bigger L/W ratio,a better transparency of endosperm and a lower percentage of chalky rice than M-1 and Pi4 b.These results showed OsBRG1 could regulate the development of seed.The expression pattern of grain related genes in M-1,M-2 and Pi4 b indicated that the expression level of GL7 in M-1 was higher when young spikelet was developed on2.0,3.0,9.0 and 16.0 cm.And M-1 was 4.8 times high as Pi4 b when young spikelet was 2 cm.The result suggested that OsBRG1 regulated seed development through GL7.2.OsBRW1 is another novel blast-resistant gene in rice.OsBRW1 was one of DEGs in Pi4 b under GUY11 infection.First,OsBRW1 mutant lines were obtained from Pi4 b using CRISPR/Cas9,and these mutants were susceptible to GUY11 7dpi with rank 4(susceptible),while Pi4 b was resistant to GUY11 with rank 1(resistant).The result comfirmed that OsBRW1 was a new balst-resistant gene in rice.OsBRW1 encoded a NBS-ARC protein with typical conserved domains NBS and CC,and its protein was subcellular localized in the cytoplasm and nucleus of N.benthamiana cells and rice protoplasts.The HR reaction of OsBRW1 further proved that OsBRW1 was resistant to GUY11.Finally,the molecular mechanism of OsBRG1's resistant ability in rice to blast was analyzed.OsBRW1 was up-regulated by SA,MeJA,ET and ABA treatment,indicating that this gene was involved in the reaction to biotic and abiotic stress in rice.OsBRW1 was up-regulated in both susceptible and resistant cultivars under the infection of GUY11,indicating that this gene participated in the immune process in rice to biotic stress.The expressing abundance of MAPK6/12,WRKY45,PR1 a,PR2,PR3,PR10 in wild type Pi4 b were higher than that in OsBRW1 mutant under the infection of GUY11,manifesting that the blast-resistant ability of OsBRW1 in rice was related to the stimulative expression of the above genes.3.G? is involved in the response of rice to the blast fungus infection.The expression pattern of G? in blast-resistant and susceptible cultivars showed that it was down-regulated in CO39 under the infection of GUY11,and up-regulated in the other 4 NILs.The result indicated G? might be involved in the resistance to blast fungus.Then the mutant d1-Pi4 b was obtained by using CRISPR/Cas9.We found that d1-Pi4 b was susceptible to GUY11 7dpi with rank 3,while Pi4 b wasresistant to GUY11 with rank 1(resistant).The result further suggested that G? was related to the blast-resistanct ability in rice.Finally,the prokaryotic expression system was employed to produce and purify protein complex of G?,which was consider as antigen to immune rabbit to get antiserum.Rice leaves inoculated with GUY11 were harvested to carry out IP analysis.112 proteins were separated from G? protein complex,among which the direct interaction between Hsp70 with G? was identified by BiFC.G? might delivery the stress signal of blast fungus through the interaction with Hsp70,and the molecular response of rice was induced and the defense reactions were caused to fight against rice blast.In summary,two novel blast-resistant genes in rice,OsBRG1 and OsBRW1,were discovered and identified in this study and their functional mechanisms were initially revealed.Whilst,OsBRG1 could also regulate the development of rice seeds.Furthermore,G? subunit was involved in the immune reaction to blast fungus infection in rice.These results should give us more insight into the resitant abilty to blast in rice and help to develop new rice varieties with broad blast-resistant ability. |
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