Differential Types Of Wheat Mutants With Hypersensitive Reaction-Like Trait To Powdery Mildew Infection And A Novel Gene Ndhrl1

Wheat hypersensitive reaction-like(HRL)mutants are characterized as the formation of necrotic lesions without infection by pathogens,and such genetic defects often lead to enhanced resistance to pathogen infection and constitutive expression of defense response genes.Chinese wheat cv.Ning7840 confers a high level of broad-spectrum resistance to rusts and powdery mildew diseases at the adult plant stage mediated by a spontaneously occurring hypersensitive reaction-like phenotype.In this study,two EMS-induced mutants of cv.Ning7840,Dllm1 and Dlm2,showed the HRL deficiency phenotype.These two mutants were susceptible to powdery mildew.At heading stage when the HRL symptoms appear on the leaves of Ning7840,the RNAs were harvested from the flag leaves of Dlml,Dlm2 and Ning7840 leaves and were sequenced to decipher the molecular mechanisms underlying HRL phenotypes.The results suggest that the diterpenoid-related stress response acts as an indirect regulator of the HRL phenotype and may be associated with broad-spectrum resistance in Ning7840.Oxidative phosphorylation,proteasome,and chlorophyll were involved in the suppression of the HRL expression in the DLM mutants.These results provide new insight into the molecular mechanism of HRL and broad-spectrum resistance in wheat.In this study,we carried out EMS mutagenesis and continuous selection of progenies,and obtained 14 mutants with more obvious HRL phenotype than the wild type.Through treating the 14 mutants and the wild type with four levels of nitrogen,the results showed that low level of nitrogen induced the early expression of HRL traits in the wild type.With the increase of nitrogen dosage,two mutants(NMu-HRL-1 and NMu-HRL-2)still showed HRL traits despite of high nitrogen condition,thus were nitrogen-independent mutants.The nitrogen fertilizer uptake and utilization efficiencies of the two types of mutants were negatively correlated with nitrogen levels.At the same nitrogen level,the panicle nitrogen fertilizer uptake and utilization efficiency of the nitrogen-independent mutant was significantly higher than that of the nitrogen-dependent mutant.Under low nitrogen condition,the chlorophyll content of nitrogen-independent mutants was significantly lower than that of nitrogen-dependent mutants.There was no significant difference in chlorophyll content between the two types of mutant plants under high nitrogen condition.Comparing the severities of powdery mildew in the 13 nitrogen-dependent(including the wild type)and the two nitrogen-independent mutants,we found that there was a significant difference in the severity of powdery mildew between the two types of mutants.The severities of powdery mildew on the nitrogen-dependent mutants were significantly higher than those of nitrogen-independent mutants under high nitrogen conditions.Nitrogen-independent mutants were still highly resistant to powdery mildew under high nitrogen conditions.In a wheat line P7001,HRL trait was expressed at low level of nitrogen,but disappeared when sufficient N was supplied,thus the phenotype was regulated by nitrogen and called it nitrogen-dependent.Using BSA strategy together with RNA-seq technique(BSR-Seq),we identified an nitrogen-dependent HRL gene(Ndhrll)and mapped it to the short arm of chromosome 2B using an F5 recombinant inbred population developed from the cross of P7001 × P216.The putative gene was located at an interval between the CAPS/dCAPS markers 7hrC9 and 7hr2dc14,and co-segregated with the dCAPS marker 7hrdc2.On the basis of this study,207 SSR primer pairs were screened between P7001 and P216 by blasting to the reference sequences of Chinese Spring.Three marks of them and a cosegregation mark 7hrdc2 were applied to identify the genotype and phenotype of 2437 plants derived from F7 secondary populations,and Ndhrll was delimited into an interval of 8.5 Mb flanked by the molecular markers 7hrdc2 and C075783.From the chromosome location and characteristics of the gene,Ndhrll is likely to be a novel gene in wheat.Further analysis of RNA-seq data showed that plant-pathogen interaction,nitrogen metabolism,zeatin biosynthesis and plant hormone signal transduction pathways were significantly differentially expressed between HRL plants and non-HRL lines.This study is of great significance for analyzing the broad-spectrum resistance mechanism and clarifying the relationship between nitrogen fertilizer,allergic reaction and disease resistance.