| Brown planthopper (Nilaparvata lugens Stal, BPH) is a monophagous pest of rice (Oryza sativa L.) and also one of the most devastating agricultural insect pests that cause rice yield loss. One of the most effective and environmentally-friendly means to control this pest is to cultivate resistant rice varieties. After several cycles of attack and counter-attack, rice and BPH formed a relationship termed co-evolutian. New biotypes of BPH emerge and overcome the genetic resistance of rice;rice evolves novel resistance forms, which in turn leads to new BPH biotypes. The emergence of new BPH biotype hindered long-lasting control of this destructive pest.To date, more than 30 major BPH-resistance genes have been identified globally, which are mainly concentrated on the rice chromosome 4,6 and 12, in form of BPH-resistant gene clusters. In particular, on the long arm of chromosome 12, eight major BPH-resistance genes were isolated independently by different research group. The relationship between these genes in clusters has aroused much interest among researchers, but because of the small number of cloned BPH-resistance genes together with the unknown sequence information in the different rice varieties making it difficult to be elucidated. Based on the cloning of BPH9 on chromosome 12 in our laborotary, in this study, we sequenced the other BPH-resistance genes on the long arm of chromosome 12 at BPH9 locus, and found that the 8 BPH-resistance genes, including BPH1, which was the earliest and most widely used gene are different or identical allelotypes of the BPH9 locus. To honor the priority in the literature, we named this locus BPH 1/9. Specifically, they can be divided into four allelotypes: BPH 1/9-1, BPH 1/9-2, BPH1/9-7, BPH 1/9-9. Wherein BPH 1/9-1 comprises BPH1, BPH10, BPH18 and BPH21; BPH1/9-2 comprises BPH2 and BPH26; BPH1/9-7 comprises BPH7; and BPH1/9-9 comprises BPH9.Sequence analysis of 117 germplasm detected 520 nucleotide polymorphisms and 21 haplotypes. This locus showed significant differentiation between indica and japonica rice group. The nucleotide sequence in the japonica rice population was much conserved (π=0.00081, and 0=0.00313),all coding for a premature protein. Analysis of the mutant sites (2041 T) leading to premature termination found that this site was G in the the wild relative species Leersia and most of the indica group; thus, it is likely the original base at this site. It mutated to T and later became fixed in the japonica group. Significant negative Tajima’s D and Fu and Li’s D values indicated that the japonica group had undergone either purifying selection or population expansion. The four resistance allelotypes were all in the indica rice population. The nucleotide polymorphisms in the population were high, and the values of Tajima’s D, Fu and Li’s D and Fu and Li’s F were all significantly larger than 0, indicating an obvious balance selection. Analysis of the corresponding coding region of the domain showed that the LRR region had a much higher nucleotide diversity than other domains (π=0.07928,0=0.05131). At the same time, sequence divergence between the resistance allelotypes were also mainly reside in the LRR domain, indicating a recognition of specificity may lead by LRR domain.Further analysis of the mechanism mediated by BPH9 revealed an important role of plant hormones. Hormones are often interacting with each other forming a complicated signal internet. In order to study the difference of hormone profiling in resistant and susceptible rice and rice varieties harboring different resistance genes (NIL-BPH6 and NIL-BPH9), we carried out a comprehensive and systematic "hormonome" analysis, which included 8 different timepoints post BPH infestation, and contain salicylic acid (SA), jasmonic acid (JA), gibberellins (GAs), auxin (IAA), abscisic acid (ABA), cytokinins (CKs) and brassinolides (BRs) in total 7 types and 26 kinds. In the NIL-BPH6, SA, JA, JA-Ile and CKs, increased substantially. In the NIL-BPH9, SA, JA and JA-Ile increased obviously. It indicates that these hormones act a major role in the two resistant varieties and also infer a different resistance mechanisms mediated by the two genes. The contents of other hormones were highly correlated, especially in resistant rice varieties, indicating that the hormones regulate the growth and defense by acting as a network. We also found that the response rate of the resistant varieties to BPH was faster than susceptible variety, which was reflected in the change of the hormone content just after BPH feeding for 1 hour, while the susceptible cultivar 9311 not only changed little, but also changed lately after 48h of BPH infestation.Based on the changes of the content and the regulation of gene, together with the study of pal and cpm2 mutants, we speculate a crosstalk model between SA and JA: an antagonistic relationship exist between the two hormones when they keep a normal level but when any of the one increased to the threshold level, they changed to an antagonistic relationship. In addition, the transgenic lines in the back of BPH6 near-isogenic lines including the inhibition of the synthesis of SA and JA, and the promoter of CK response regulator OsRR6, were generated to study the roles of the three hormones in the mechanism of resistance mediated by BPH6. Up to now, we have obtained T1 generation of transgenic lines. The follow-up phenotypic identification will be carried out soon.The allelic diversity analysis of BPH1/9 locus will provide the theoretical guidance for coping with the new biotype of BPH. Subsequent mining of the other resistant alleles and artificial shuffling of the existing allele will help long-lasting and effectivly control of the destructive pest. The hormonone analysis in resistant and susceptible rice varieties after BPH infestation laid the foundation for elucidating the interaction among the hormone network in rice and the resistance mechanism mediated by different BPH resistance genes. |
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