Functional Characterization Of Nitrogen-Dependent Hypersensitive Reaction Like Gene Ndhrl1 And Mapping Of Its Inhibiting Gene Ihrl1 In Wheat

Hypersensitive reaction like（HRL）refers to the lesion-like symptoms that small yellowish or brown spots appeared randomly and dispersedly on all the leaves in the absence of plant pathogens.Most of the genes that control HRL traits are involved in programmed cell death（PCD）and enable plants to have durable and broad-spectrum disease resistance.The most economical and effective way to control plant diseases was to apply such resistance genes,which serves as an attractive target for studies on the regulation of cell death.Our previous studies have shown that the wheat line P7001 showed hypersensitive reaction-like（HRL）trait at low nitrogen supply,and this trait was controlled by a dominant gene Ndhrll（Nitrogendependent hypersensitive reaction-like 1）.Ndhrll was the first reported nitrogen-dependent HRL gene,and located in the 13.5 kb region of 2BS chromosome by map-based cloning,which contained only one complete gene.On this basis,we carried out the following work in this study:1.A two-factor experiment of nitrogen gradient × genotype was designed for the nearisogenic lines 1618HRL and 1618non-HRL.Variance analysis showed that Ndhrl1 gene had no significant effect on spike number and yield of wheat.The analysis of Ndhrl1 expression pattern showed that the gene expression was increased gradually from seedling to heading stage,reaching a high level at booting stage and heading stage,and the expression of Ndhrl1 was induced by nitrogen starvation.2.The wheat line Bobwhite without HRL phenotype was used as transgenic receiver material to carry out the complementary verification experiment of Ndhrl1.The results of Trypan blue staining and DAB staining indicated that the HRL phenotype of the complementary plants may be caused by the accumulation of reactive oxygen species（ROS）that promote the cell death of the leaves,suggesting that Ndhrl1 mediated the HRL phenotype.3.Combined with the whole-genome reference sequence of wheat cv.Chinese Spring（CS）,we designed primers for Ndhrl1,and found that there were 4 synonymous mutations and 2 missense mutations in the coding region of Ndhrl1 between the two parents,and 3 mutation sites in the promoter region.In order to study whether the variation of the coding region of Ndhrl1 is related to the HRL phenotype,we constructed the overexpression vector of ndhrl1,and carried out agrobacterium-mediated genetic transformation.149 regenerated seedlings were obtained,but no positive transgenic seedlings were obtained.Considering that there are three variation sites in the promoter region of Ndhrl1,and the expression of Ndhrl1 is significantly different between the two near-isogenic lines,which suggested that the mutation in the promoter region may be related to the regulation of the expression of Ndhrl1.In order to study the functional sites regulating the expression of Ndhrl1 in the promoter region,we constructed a series of expression vectors containing different variation sites and their combinations.PCR verification and sequencing results showed that the vector construction was successful.4.In order to investigate the robustness of the genetic effect of Ndhrl1 under different genetic background,seven genetic populations,with P7001 being the common parent,were constructed and analyzed.Six of the seven populations（F2）showed a segregating ratio of HRL:non-HRL=3:1.Unexpectedly,different from the other six combination,the F1 plants of P7001/Fielder showed non-HRL trait and the F2 plants from the cross of P7001/Fielder showed the contrasting segregation ratio of HRL:non-HRL=1:3.It was interesting that Fielder carried the same Ndhrl1 gene as P7001 by sequencing.We speculated that Fielder may carry an inhibiting gene of Ndhrl1.Using 55 K SNP array and PCR-based markers,the inhibitory gene,designated Ihrl1（Inhibitor of hypersensitive reaction-like）,was mapped to an interval of 5.63-12.91 Mb on the short arm of chromosome 2B.Interestingly,Ihrl1 is tightly linked to Ndhrll and functions by inhibiting hypersensitive reaction-like cell death to suppress disease resistance mediated by Ndhrl1,and may be also involved in nitrogen metabolic and（or）signaling pathways.In summary,we verified the function of Ndhrl1,mapped the inhibiting gene Ihrl1,and elucidated their genetic relationship.These results would further help decipher the relationships among nitrogen fertilizer,hypersensitive reaction and plant disease resistance.