| Rice is the most important food crop in the world.Rice blast disease is one of the main diseases of rice.The occurrence of rice blast disease can cause rice production to be reduced sharply,and even crop failure.It is important to study and concise the rice blast resistance mechanism for rice blast resistance breeding.In this paper,the resistance to rice blast was identified by Magnaporthe grisea inoculation method firstly,and the resistance and susceptible to rice blast varieties were selected.The composition of rice blast resistance major genes Pi-b,Pi-ta and Pi-k in 15 tested rice materials was detected by PCR.Further,the resistance variety Zhongyul4 and susceptible variety to rice blast zhongjiangjing31450,which both contain Pi-b,Pi-ta,Pi-k genes,were selected as subsequent experimental materials.The response of Pi-b,Pi-ta and Pi-k gene expression to environmental factors,hormones,Si and H2O2 treatment was analyzed by qRT-PCR.The response of genome-wide transcription and protein expression to the Magnaporthe grisea treatment was analyzed by using transcriptome sequencing,qRT-PCR and proteomics techniques.The main results are as follow:1.Response of rice blast resistance genes to environmental factors,hormones,Si and H2O2Low-temperature and weak-light treatment resulted in significant up-regulation expression compared with control of Pi-b in both rice blast resistant and susceptible varieties,but high-humidity had no significant effect on the expression of Pi-b;Low-temperature,weak-light and high-humidity treatment all led to significant up-regulation expression compared with control of Pi-ta and Pi-k in rice blast resistant variety Zhongyul 4 leaves.Weak-light treatment also led to significant up-regulation expression compared with control of Pi-ta and Pi-k in rice blast-susceptible variety zhongjiangjing31450 leaves.The expression of Pi-k and Pi-ta were significant up-regulation compared with control by the treatment of six hormones,Si and H2O2 in rice blast resistant variety zhongyul4 leaves.Meanwhile,the expression of Pi-ta was also significant up-regulation compared with control by the treatment of KT,paclobutrazol and Si in rice blast susceptible variety zhongjiangjing31450 leaves.But the effect of the above treatments on the expression of Pi-k in rice blast susceptible variety zhongjiangjing31450 leaves was not obvious.The expression of Pi-b was significant up-regulation compared with control by the treatment of ABA?KT?IAA in rice blast resistant variety zhongyul4 leaves;The expression of Pi-b was also significant up-regulation compared with control by the treatments of Si,KT,paclobutrazol,in rice blast susceptible variety zhongjiangjing31450 leaves.In summary,the expression of Pi-ta,Pi-b and Pi-k were induced by adverse environmental factors of low-temperature,weak-light and high-humidity,and also induced by six hormones,Si and H2O2 treatments.Compared with the two tested varieties,the degree of expression induction of Pi-ta,Pi-b and Pi-k to various factor treatment in rice blast resistant variety was higher than that in rice blast susceptible variety.This may be one of the important causes leading to the resistant difference to rice blast between the two rice varieties.2.Rice gene transcription response to Magnaporthe grisea treatmentThe results of RNA-seq indicated that the gene expressions including antibacterial,secondary metabolites,detoxifying enzymes,antioxidant enzymes and chaperone proteins,transcription factors,hormone metabolism and response-related genes in two tested rice leaves changed under the treatment of Magnaporthe grisea.(1)Antibacterial-related genes only specifically expressed inducibly in blast-resistant rice variety zhongyul4 by the treatment of Magnaporthe grisea included putative disease resistance RPP13-like protein 3,protein LURP-one-related 5,protein LURP-one-related 7,protein TIFY 10c,ent-cassadiene C11-alpha-hydroxylase 1,defensin-like protein,chemocyanin,thaumatin-like protein,transmembrane protein 45B and momilactone A synthase-like.In particular,putative disease-resistant RPP13-like protein 3 that was homologous to rice blast-resistant gene Pi-ta,momilactone A synthase-like,defensin-like protein,thaumatin-like protein gene were all inducibly expressed by the treatment of Magnaporthe grisea,which might render the high blast-resistance to zhongyul4 variety.The results also indicated antibacterial-related genes that were only specifically inhibition in rice blast-susceptible variety Zhongjiangjing31450 by the treatment of Magnaporthe grisea.In particular,the expressions of putative disease resistance RPP13-like protein 3 which was homologous to Pi-ta and RPM1-interacting protein 4 were inhibited by the treatment of Magnaporthe grisea which might contribute highly susceptivity to this rice variety.(2)There were 11 secondary metabolites-related genes were inducibly expressed in zhongyul4 by the treatment of Magnaporthe grisea,however,only 2 secondary metabolites-related genes were inducibly expressed in zhongjiangjing31450.These results indicated that the treatment of Magnaporthe grisea to a greater extent activated the secondary metabolism in blast-resistant varieties than in blast-susceptible varieties,which might confer zhongyul4 upon the high blast-resistance.(3)In blast-resistant variety zhongyu 14 leaves,quite a few Phase ? associated cytochrome P450,Phase ? detoxification associated genes,such as glutathione S-transferase GSTU6 and UDP-glycosyltransferase,and 5 Phase ? detoxification associated ABC transporter family member genes were inducibly expressed by the treatment of Magnaporthe grisea.However,the expressions of glutathione S-transferase GSTU and ABC transporter family member genes were inhibited by the treatment of Magnaporthe grisea in hongjiangjing31450.In addition,the expressions of detoxification protein 35 and 29 genes were induced by the treatment of Magnaporthe grisea in zhongyu 14 leaves,On the contrary,the expressions of detoxification protein 21,48,and 49 genes were inhibited by the treatment of Magnaporthe grisea in blast-susceptible variety zhongjiangjing31450.These results suggested that under the treatment of Magnaporthe grisea,the blast-resistant variety had stronger detoxification capacity than the blast-susceptible varieties.(4)The expressions of transcription factors-related genes including dehydration-responsive element-binding protein 1G,ethylene-responsive transcription factor ABR1,5 WRKY transcription factors,NAC transcription factor 29,NAC domain-containing protein 92 and MADS-box transcription factor 47 were all induced by the treatment of Magnaporthe grisea in blast-resistant variety zhongyul4 leaves.On the contrary,the expressions of quite a few dehydration-responsive element-binding proteins,ethylene-responsive transcription factor,WRKY transcription factor,transcription factor bHLH,NAC domain-containing protein and transcription factor MYB were inhibited by Magnaporthe grisea in blast-susceptible variety zhongjiangjing31450 leaves.It is easy for us to infer that these genes are important for zhongyul4 with stronger blast-resistance and zhongjiangjing 31450 with lower blast-resistance.(5)The expressions of genes including 1-aminocyclopropane-l-carboxylate oxidase homolog 1 catalyzed ethylene synthesis,gibberellin 2-beta-dioxygenase 8 inhibited the active gibberellin conversion,cytochrome P450 94B3 negatively feedbacked and controled JA-Ile level and auxin-responsive protein SAUR36 regulated leaf senescence were all induced by Magnaporthe grisea in blast-resistant variety zhongyul4 leaves.On the contrary,all these genes were inhibited in blast-susceptible variety zhongjiangjing31450 leaves.Additionally,more genes involved in hormone metabolism and response pathway were inhibited Magnaporthe grisea in blast-susceptible variety zhongjiangjing31450 leaves.These results would help us to understand,from plant hormone metabolism,why zhongyul4 was highly resistant to rice blast disease and zhongjiangjing31450 highly susceptible.3.Proteomic response to Magnaporthe grisea treatment in riceProteomic analysis results indicated that the expressions of antioxidases(including superoxide dismutase and 2-Cys peroxiredoxin BAS1),photosynthesis-related enzymes or proteins(including Ru Bis CO large subunit and ferredoxin-1)and enzymes related to amino acid metabolism(including glycine cleavage system H protein,glycine dehydrogenase and glutamine synthetase)were all increased expression under the treatment of Magnaporthe grisea in blast-resistant variety zhongyu14 leaves,which conferred blast-resistant rice upon the powerful defense against peroxidation,the higher photosynthesis and quicker amino acid synthesis and metabolism upon the rice blast infection.Inducible expressions of several HSPs was the major features of the proteomic response to the treatment of Magnap orthe grisea in blast-susceptible variety zhongjiangjing31450 leaves.In addition,the antibacterial related Barwin and nsLTP 1 and detoxification related protein glutathione S-transferase GSTF1 were induced by the treatment of Magnaporthe grisea in blast-susceptible variety zhongjiangjing31450 leaves.Quite a few protein expressions were inhibited by the treatment of Magnaporthe grisea in zhongjiangjing31450 leaves which included proteins involved in glycolysis and electron transport in the respiratory chain,proteins associated with photosynthesis,proteins associated with proteolysis and transport,proteins associated with transcriptional regulation and initiation,proteins associated with antibacterial(protein P21),and so on.The down-regulated expression of these important proteins would reduce the energy supply needed for disease resistance in blast-susceptible rice variety,reduce the rotation of the protein in the leaves,and suppress the expression of resistance-related genes.These changed gene expression profile might be closely related with the susceptibility of zhongjiangjing31450 to rice blast disease. |
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