| Rice is one of the most important food crops.In the main cultivation area of rice,brown planthopper(BPH)is the most destructive pests to rice.BPH(Nilaparvata lugens Stal)is a monophagous,phloem-sucking herbivore.It sucks the sap from the rice phloem using its stylet,causes direct and indirect damage to rice growth,seriously,even causes rice death.The most economical and environmentally friendly method is to breed resistant rice varieties to against BPH attacks.Since the first BPH resistance gene BPH1 was identified from 1969,more than 30 BPH resistance genes have been genetically defined,most of which are from cultivated rice(e.g.Mudgo,ASD7,Rathu Heenati)and wild rice(e.g.Oryza officinalis,Oryza minuta,Oryza latifolia).The objective of this study is the candidate gene of BPH15 resistance gene,named LecRK.BPH15 derives from wild rice(Oryza officinalis).It was mapped on the short arm of rice chromosome 4 and exhibits high resistance to BPH.Receptor-like kinase LecRK exists widely in higher plants,whose structure can be divided into three parts,extracellular lectin domain,transmembrane domain and intracellular kinase domain.It has been reported that LecRK was involved in the regulation of plant development,responding for hormonal signaling pathway and biotic stress and abiotic stress.In this study,we explored the function of LecRK by reverse genetics.The resistance mechanism was preliminarily explored and the experimental results are as follows:1.Subcellular localization experiments confirmed that LecRK is a membrane targeting protein.After the susceptible rice Kasalath was fed by BPH,the expression of LecRK could be induced,as well as the response of PTI marker genes.Then,LecRK overexpression and inhibited expression vectors were constructed and transformed into the BPH-susceptible rice variety Kasalath.The overexpression and inhibited expression homozygous T2 transgenic lines were selected for BPH-resistance and disease-resistance evaluation.Host choice tests revealed that BPH was more likely to choose LecRK inhibited expression plants,followed by wild type plants,and finally LecRK overexpression of plants,suggesting that LecRK do act via antixenosis.BPH growth weight and honeydew excretion tests revealed that BPH insects feeding on LecRK inhibited expression plants produced significantly higher BPH growth weight and honeydew excretion than those on WT plants,Conversely,BPH growth weight and honeydew excretion produced by BPH Insects feeding on LecRK overexpression plants showed no significant differences compared with those on WT plants.These suggested that the resistance of LecRK inhibited expression plants to BPH was the weakest,while the resistance of of LecRK overexpression plants was the strongest,which indicated that LecRK played a positive regulated role for the resistance of rice to BPH.Experimental results of BPH mortality rate showed that there was no significant difference in the mortality rate of the BPH in LecRK overexpression plants or in the inhibited expression plants when compaired with that in WT plants,which indicated that LecRK gene expression was not lethal to BPH.A leaf cutting inoculation method was used in LecRK transgenic plants to examine the resistance to bacterial blight.The LecRK inhibited expression plants show increased susceptibility to bacterial blight than WT plants,while The LecRK overexpression plants show increased resistance to the disease than WT plants.Those results above indicated that LecRK was involved in both insect resistance and disease resistance.We hypothesized that LecRK should activate the PTI response in rice,although the resistance is not so strong as that of ETI,it may be a broad-spectrum resistance,which is involved in insect resistance and disease resistance.2.After the rice protoplasts expressed LecRK is treated with BPH and chitin powder,the expression level of LecRK was higher than that of untreated group.Additionally,the expression of PTI marker gene in treatment group was higher than that in untreated group.These results showed that both BPH treatment and chitin treatment could induce the expression of PTI marker genes,and the expression of PTI marker gene was up-regulated more in the rice protoplasts expressed LecRK.After chitin treatment,the protoplasts prepared from LecRK inhibited expression plants and WT plants both showed ROS burst,and the ROS level of the former is lower than tha of the latter.The results indicated that the ROS induced by chitin was inhibited along with the inhibitation of LecRK expression3.By yeast two hybrid system,we use extracellular domain of LecRK to screen the BPH library in order to identify some putative PAMP molecules in BPH.After two rounds of screening,we obtained 79 candidate interacting proteins,from which we selected 6 candidate proteins for further validation and investigation.By one-to-one verification in yeast cells and CoIP confirmation in rice protoplasts,we found these 6 candidate proteins could interact with both LecRK extracellular domain and full length.The protein expression of LecRK was not affected by the co-expression of these candidate proteins with LecRK.It is suggested that the expression of LecRK was not regulated by the candidate protein.Besides,we found these candidate proteins also had no effect on the phosphorylation status of LecRK by Phos-tag experimental method.4.The BPH3 gene found in RH material is the same as the three LecRK sequences found in our laboratory in B5 rice variety.In order to explore the relationship between the three LecRKs in B5 and the LecRK in this paper,the three LecRK sequences were amplified from B5 cDNA,and their coding proteins were verified to interact with themselves and each others.We found these four LecRK could associate with chitins,so we speculated that these four LecRK may exercise the function of the PRR molecule,which can identify chitin and stimulate the downstream signaling pathway.5.MicroRNA is a kind of small molecule RNA which widely existing in animals and plants.Although their amounts are not much,but they play a very important rolein regulation.From the perspective of microRNA,we analyzed the mechanism of insect resistance of BPH15.MicroRNA,which may be involved in the resistance response to BPH,was found to be involved in the regulation of hormones,resistance and signal transduction.Additionally,some microRNAs which act as the key regulatory roles in resistant materials were analyzed.Also,the target genes of these microRNAs were predicted and identified... |
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