Study On The Mechanism Of AMT1-Mediated Ammonium Uptake Regulating Rice Resistance To Sheath Blight

Rice sheath blight caused by Rhizoctonia solani Kühn（R.solani）is the disease with the largest incidence and control area in rice production,which seriously threatens rice production.However,the genetic resources which highly resistant to sheath blight,and the research on the disease resistance mechanism are limited.Nitrogen fertilizer is of great significance in promoting the growth,development and yield of crops.However,nitrogen application has been reported to affect the occurrence of plant diseases.Previous studies have shown that nitrogen uptake and metabolism are correlated with plant diaease resisatnce,but there is no relevant molecular evidence.In this study,rice sheath blight was selected as the research object,and the key gene mutants of ammonium uptake and metabolism pathway were used to explore the function mechanism of ammonium uptake and metabolism in rice defense against sheath blight.The main results are as bellow:1.Identification and functional analysis of AMT1;1 D³⁵⁸N mutantIn this study,a mutant more susceptible to sheath blight was identified in the Ds insertion rice mutant library,but no Ds insertion was detected in the mutant.Considering the mutant has a pale green leaf phenotype,the genes involved in chlorophyll biosynthesis were cloned and sequenced.Among the genes examined,we found that the G¹⁰⁷²was changed to A at the AMT1;1 conding region,resulting the aspartic acid³⁵⁸changes to asparagine(D³⁵⁸N),and the mutant was named AMT1;1 D³⁵⁸N.Analysis of conserved amino acid residues and their function in AMT1;1 ammonium transport showed that D³⁵⁸was highly conserved among plant AMTs,and D³⁵⁸N mutation abolished NH₄⁺uptake activity of AMT1;1.2.AMT1;1 D³⁵⁸N interacts with AMT1s and inhibit their ammonium transporting function to negatively regulate the resistance of rice to sheath blight.The results of resistance identification and NH₄⁺content determination showed that compared with the wild type（WT）,the rice with overexpression of mutant AMT1;1(AMT1;1D³⁵⁸N)has less NH₄⁺accumulation and is more susceptible to R.solani.However,although silencing AMT1;1 inhibited the NH₄⁺uptake in rice,it had no effect on rice resistance to sheath blight.The mechanism analysis based on split ubiquitin yeast two-hybrid,bimolecular fluorescence complementation and ammonium transport activity test confirmed that AMT1;1D³⁵⁸N interacts with AMT1;1,AMT1;2 or AMT1;3 and inhibit their ammonium transporting function to negatively regulate the resistance of rice to sheath blight.3.AMT1;1 is a potential target gene with high nitrogen use efficiency,high disease resistance and high yield under low nitrogen conditionsThe results of inoculation and NH₄⁺uptake efficiency of AMT1;1 OX and AMT1 RNAi plants（AMT1;1,AMT1;2 and AMT1;3 are silenced）confirmed AMT1;1-mediated NH₄⁺uptake positively regulates rice sheath blight resistance and nitrogen use efficiency（NUE）.The transgenic rice,p Os AMT1;1-At AMT1;3T464D-A141E Amtrac,with expression of high capacity-ammonium sensor driven by Os AMT1;1 promoter was further constructed.NH₄⁺content determination and R.solani inoculation results showed that improving ammonium uptake activity enhances the resistance of rice to sheath blight.Further analysis of AMT1;1OX and AMT1 RNAi plants cultured under different nitrogen conditions found that overexpression of AMT1;1 can enhance the resistance to sheath blight and NUE of rice under low and medium nitrogen conditions,but not under high nitrogen condition.The results of yield determination showed that overexpression of AMT1;1 increased the 1000-grain weight of rice under low nitrogen conditions,suggesting that AMT1;1 is a potential target gene with high NUE,high disease resistance and high yield under low nitrogen conditions.4.AMT1;1 mediated NH₄⁺uptake regulates rice resistance to sheath blight by promoting the accumulation of nitrogen metabolites rather than directly regulating NH₄⁺contentThe results of disease resistance identification and NH₄⁺content determination of the mutant（gs1;1）and overexpression plants（GS1;1 OX）of GS1;1,the key gene of nitrogen metabolism,and plants with overexpression of AMT1;1 in the gs1;1 background（AMT1;1OX/gs1;1）confirmed that nitrogen metabolism rather than NH₄⁺itself was necessary for rice sheath blight resistance.Further analysis based on physiological and genetic experiments found that AMT1;1 triggered nitrogen metabolism enhance the resistance of rice to sheath blight by increase the content of amino acid and chlorophyll,but not of GABA signal.5.AMT1 forms a feedback regulatory cycle with ethylene signaling regulating the NH₄⁺uptake and then enhancing rice resistance to sheath disease under low nitrogen conditionsAnalysis of downstream regulation mechanism of AMT1 mediated NH₄⁺uptake based on transcriptome sequencing found that ethylene signaling related genes were abundant.Previously,our group identified the positive regulation of ethylene signal on rice sheath blight resistance.In this study,the expression level of several ethylene biosynthesis and signal genes were significantly induced by NH₄⁺.Moreover,the expression of typical positive regulatory factors of ethylene signaling in AMT1;1 OX and AMT1 RNAi plants cultured under low nitrogen condition are significantly indued and decreased,respectively,while the expression of negative regulatory factors is on the contrary.However,in high nitrogen condition,the expression of ethylene signal positive and negative regulators in the three plants has no obvious regularity.It indicates that AMT1;1-mediated NH₄⁺uptake enhances the rice resistance sheath blight through activing ethylene signal.In addition,ethylene signal can feedback regulate the ammonium-dependent induction of AMT1,revealing a new mechanism of feedback regulation formed between AMT1;1 and ethylene signal to regulate NH₄⁺uptake to enhance rice resistance against sheath blight under low nitrogen condition.In conclusion,based on the identification and functional analysis of mutant of ammonium transporter AMT1;1,this study explored the mechanism of AMT1;1 D³⁵⁸N inhibiting the ammonium transport activity of AMT1 by interacting with AMT1;1,AMT1;2and AMT1;3,and then inhibiting the resistance of rice to sheath blight.This study also revealed the potential value of AMT1 in improving rice resistance,NUE and yield under low nitrogen conditions and the mechanism of AMT1;1 improving rice resistance to sheath blight by promoting nitrogen metabolism and ethylene signaling.The results will broad a new perspective for the study of the relationship between nitrogen and sheath blight and the mechanism of rice sheath blight resistance at the molecular level.This study will provide target genes for the molecular improvement breeding of crops with high NUE,high yield and high resistance under the background of"double reduction".It also provids a new direction for the precise use of nitrogen to increase rice yield and improve the immunity of rice to sheath blight as well as other pathogenic diseases.