| Rice sheath blight is one of the three major rice diseases,which commonly occurs in rice cultivating area in China,and severely affect rice production up to 50%.Chemical control is still main approach to protect rice from sheath blight disease,but chemical fungicide overwhelmingly pollute the environment,and the problem of pathogens' resistance to fungicide is becoming more and more serious,which brings great difficulties to the control of rice sheath blight.Resistance breeding is an economic,eco-frined and effective solution,but the researches regarding defense mechanism of rice sheath blight resistance are fewer,also,it lacks resistant genes for resistant breeding.Current genetic resources are poor and can not be applied to production directly.Therefore,isolation of sheath blight disease resistant genes became urgent.In our preliminary transcriptom study,we identified two NAC transcription factor members NAC90-1 and NAC90-2 were induced upon inoculation of Rhizoctonia solani.Further,we focused on NAC90 performed a serease of experiments,and the results obtained are shown bellow:1.Total RNA was extracted from rice leaves after 0,24,48 and 72 hours of R.solani AG1-IA inoculation.The c DNA was synthesized via reverse transcription gene expression was determined by real-time quantitative PCR.The results showed that the expression levels of XNDLs were induced by R.solani AG-1 IA.Furthermore,amino acid sequence alignment and phylogenetic tree analysis were carried out,and we found that NAC90 and At XND1 were highly conserved;therefore,NAC90 was named XND-Like(XNDL).Subcellular localization of XNDLs shows that XNDL1-GFP was localized at the nucleus and plasma membrane,while XNDL2-GFP is localized in the nucleus.Yeast mono-hybrid test was conducted to verify whether XNDLs have transcriptional activation activity,and it was found that full length and C terminal of XNDL1 and 2 have transcription activation activity in yeast.2.To investigate XNDLs function duringrice resistance to sheath blight,XNDL1 and 2 CRISPR/Cas9-mediated genome editing mutants as well as XNDL2 overexpressors were generated.Sequencing results indicated that xndl1-1 and xndl1-2 are base adding and deletion mutants,respectively,while xndl1-1 and xndl1-2 are base deletion and addition mutants,respectively.In XNDL2 overexpressors,XNDL2 expression level was obviously higher than in wild-type control.Inoculation of R.solani AG1-IA indentified that xndl1 and xndl2 mutants are more susceptible whitl XNDL2 ovreexpressors are less susceptible to sheath blight disease compared to wild-type control,suggesting that XNDLs positively regulate rice resistance to sheath blight disease.3.XNDLs positively regulate rice resistance tosheath blight disease,but the underlying defense mechanism remians unclear.To understand XNDLs regulatory mechanims,transcriptome sequencing was carried out using wild type control and XNDL2 overexpressor plants.Compared to wild-type control,518 differentially expressed genes XNDL2 overexpressor.Among them,358 genes were up-regulated while 160 genes were down-regulated in XNDL2 overexpressor than in wild-type control.Nine differentially expressed genes including EIN2 ? EIL2 ? RGA3 ? RGA4 ? RPM1 ? RPP13-LIKE1 ? RGA4-LIKE ? RGA4-2 ?LOC4341545were verified by q PCR and found that the expression patterns were similar with transcriptome data.Further,518 diffrentially expressed genes were futher classdied into GO and KEGG terms.GO wasmainly concentrated in binding and catalytic activity,and that the cellular components were mainly concentrated in organelles and cell membranes.The main biochemical metabolic pathways and signal transduction pathways of KEGG are Toll-like receptor signaling pathway and phenylpropanol biosynthesis;EIN2 was identifed from transcriptome results,and which is the key regulator of ethylene signal,and its mutantein2 showed more susceptible syptome than wild-type control,suggesting that XNDL transcription factor might regulate rice resistance to sheath blight partly via activation ethylene signal. |
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