Isolation And Functional Characterization Of Pathogen-induced Defense-responsive Genes Of Rice

Rice bacterial blight disease caused by Xanthomonas oryza sative pv.oryza and fungal blast disease caused by Magnaporthe grisea,are two of the most devastating diseases of rice worldwide。These two diseases cause tremendous yield loss each year. Efficient control of disease through improving rice defensive system is economic and green way.Characterizing rice disease resistance related genes and elucidating the mechanism of rice disease have important significance in science and application for improving rice.The signaling induced by the plant growth hormone auxin is generally recognized to regulate plant growth and development.Here we report that rice GH3-8,an auxin-responsive gene functioning in auxin-dependent development,activates disease resistance in a salicylic acid- and jasmonic acid-signaling-independent pathway.Bacteria induce accumulation of indole-3-acetic acid(IAA),the major type of auxin,in rice.IAA induces the expression ofα- andβ-expansins,the proteins that are known to loose cell wall,the native barrier of biotic intruder,to facilitate the growth of cells.In resistance rice variety carrying Xa21 or Xa26,the infection of bacteria induce rice to synthesize GH3-8 in infection site of rice.GH3-8 encodes an IAA-amino synthetase that prevents free IAA accumulation and looseness of cell wall.Overexpression of GH3-8 results in enhanced disease resistance and retarded growth and development,which is at least partly due to inhibiting the expression ofα- andβ-expansins via suppressing auxin signaling.Here we show the mechanism of bacteria hijacks auxin as virulence factor to infect rice,and the regulating pathway of rice to the virulence factor;in addition to,explain the cause that plants growth was restrained in disease resistance.Overexpression of GH3-8 results in sterility of plants.Analysis of forward cross and reverse cross showed that GH3-8-overexpressing plants were male sterility and female sterility.We found that the stigmas of GH3-8-overexpressing plants are abnormal by morphological observation.We observed the mature embryo sac of GH3-8-overexpressing plants using laser scanning confocal microscopy.The result showed that the mature embryo sacs of GH3-8-overexpressing plants were abnormal.This may be the reason of female sterility.No obvious difference was observed in stamen between GH3-8-overexpressing plants and wide type in stamen,but the most of pollen of GH3-8-overexpressing plants were sterile.This may be the reason of male sterility. GH3-8 had high expression level in stamen,and the expression of GH3-8 changes as the development of flower.Tissue and time differential expression confirmed the role of GH3-8 in regulating flower development.We gained several auxin responsive factors (ARFs) that interacted with the promoter of GH3-8 by analysis of yeast one hybrid. Overexpression of OsARF8 in Mudanjiang 8 activated the expression of GH3-8.This result suggested that OsARF8 is the transcription factor in regulating the expression of GH3-8.OsARF8 had high expression level in pistil,and very low expression level in stamen.GH3-8-overexpressing plants had lower fertility than wide type.The most of pollen of OsARF8-overexpressing plants were sterile.The overexpression of Auxin signaling genes(OsARF8 and GH3-8) resulted in decrease of rice fertility.This result suggested that auxin plays a critical role in regulating flower development.The detection of the auxin distribution in panicle development showed that auxin is affinitive relation with panicle development.The transgenic plants with repressed expression of OsDR8 showed reduced resistance or susceptibility to Xanthomonas oryzae pv.oryzae and Magnaporthe grisea causing bacterial blight and blast,respectively.The putative product of OsDR8 was highly homologous to an enzyme involved in the biosynthesis of the thiazole precursor of thiamine.Exogenous application of thiamine could complement the compromised defense of the OsDR8-silenced plants.The expression level of several defense-responsive genes including the earlier functional genes of defense transduction pathway,OsPOX and OsPAL,and the downstream genes of the pathway,OsPR1a,OsPR1b,OsPR4,OsPR5 and OsPRIO,was also decreased in the OsDR8-silenced plants.These results suggest that the impact of OsDR8 on disease resistance in rice may be through the regulation of expression of other defense-responsive genes and the site of OsDR8 function is on the upstream of the signal transduction pathway.In addition,the accumulation of thiamine may be essential for bacterial blight resistance and blast resistance.We found a mutant with lesion mimics on the leaves through selecting a T-DNA insertional rice pool.The inserted T-DNA was inserted into the open reading frame(ORF) of a gene named OsDR9.The predicted encoding product of OsDR9 consists of 180 amino acids that is an unknown functional protein.OsDR9 had very low expression level in the stem and young panicle but higher level in seedling,flag leaf,sheath and callus;no OsDR9 expression was detected in the root.In addition,OsDR9 had higher expression level in old leaf than young leaf.The mutant was highly resistance to Magnaporthe grisea causing fungal blast disease and bipolaris oryzae causing Cochliobolus miyabeanus disease in field.Histochemical detection and DNA fragmentation of the leaves developed lesion mimics showed that the cell death had the same features of apoptosis.In addition, the expression of pathogenesis related(PR) proteins genes PR4 and PR8 as well as a blast resistance related gene AOS2 was upregulated in the mutant.The mutant also accumulated autofluorescent materials,salicylic acid and phytoalexins(both momilactone A and sakuranetin).The mutant contained elevated levels of superoxide and H₂O₂.A 10.5-kb fragment harboring the OsDR9 gene from rice variety Nipponbare was transferred into the mutant.Lesion mimic phenotype was disappeared in the transgenic plants,indicating that knockout of OsDR9 by T-DNA insertion caused the lesion mimic mutant phenotype.These results suggest that OsDR9 is a negative regulator in rice disease resistance and apoptosis.