Responsive Genes Elicited By Activator Protein Isolated From Alternaria Spp. And Their Roles In Plant Disease Resistance

Elicitors from diverse plant-pathogen interaction systems were components that induce multiple defense response but have no direct toxicity to plant pests;they have showed good prospects in biocontrol of plant pests. A purified elicitor from Alternaria spp.. which have showed good properties of growth promotion and disease resistance., was selected, and an array of studies were conducted.Rice seedlings sprayed with activator protein, a novel proteinaceous elicitor originated from Alternaria spp., developed increased resistance to rice blast after 5 days. The rapid accumulation of active oxygen species H₂O₂ and O₂^- was observed in the early stage following stimulation with elicitor, however since the 7th day after treatment the contents of both kinds of AOS in unelicitored plants were higher than those in elicitored plants. Enzymatic activities of phenyalanine ammonialyase (PAL), 1,3-β-glucanases and chitnases in rice leaves were enhanced in a similar manner, arriving at a peak on the 7th, 5th and 9th day respectively. The protein also led to the transcription activation of crucial signaling pathway components NPR1 and EIN2 since the 1st day, transcription repression of CTR1 after 3 days, but had no effect on transcription level of defense gene PR4, which seemed to indicate that salicylic acid ( SA) and ethylene (Et) signal pathway may mediate the resistance plant activator protein induced.Transcriptional activation of a set of defense-related genes was crucial in plant systematic acquired resistance pathogen and other elicitors induced. In order to establish a full gene expression profile of plant activator protein gave rise to, we employed cDNA microarray analysis of 10,000 clones randomly selected from rice cDNA library. In this experiment, after treating with 2 fig ml^-1 elicitors, 136 genes were significantly induced and 93 genes were repressed. Of the 229 genes, 15 genes were possibly involved in signal transduction, 13 genes were related to disease resistance and defense. Apart from 117 unknown genes in function, others were respectively implicated in transcription, cell growth and division, cellular transportation, protein synthesis, primary and secondary metabolism, energy production and cell conformation et al..The statistic results revealed that plant activator protein induced expression of defense- and signaling-related genes such as regulatory protein NPR1, ascorbate peroxidase, cytochrome P450, Sgt1 transcriptional factor, bZIP transcription factor, tyrosine phosphatase, serine/threonine protein kinase and thiosulfate sulfurtransferase et al.. Semi-quantitative PCR and Northern blotting analysis of 10 defense- and signaling-related genes (designated as OsAiDGl-10 in turn) confirmed the microarray results except that the expression pattern of OsAiDG8 was contrary to chip results and the expression of OsAiDG1 at transcriptional level was nearly the same as the control.Protein kinase, which is nearly isolated from all organs and tissues in plants, plays an important role in regulation of signal transduction pathways of different stresses and hormone.Two protein kinase genes, designated as OsAiDG3 and OsAiDG4, were cloned by RT-PCR from total RNA extracted from rice leaves. OsAiDG3, which has high homogenous to certain known calmodulin-domain protein kinases (CDPK), encodes putative 420-amino-acid kinase with a mol .wt of 47.2 kDa and a pi of 5.02 as indicated. OsAiDG4, which has high homogenous to certain known receptor-like protein kinases (RLK), encodes putative 509-amino-acid kinase with a mol .wt of 57.1 kDa and a predicted pi of 7.74. The recombinant plasmid pGEX-6P-l- OsAiDG3 and pGEX-6P-l- OsAiDG4 was constructed respectively, and then was transformed into E.coli strain ER2566. After a four-hour induction of 0.5 mmol L/'lPTG, the two fused proteins were highly expressed with the analysis of SDS-PAGE.To better understand the possible function of the two protein kinase genes OsAiDG 3 and OsAiDG4 in rice blast resistance, Northern blotting analysis were employed to detect their expression in different rice cultivars of Yuanfenzao and 1312 and before and after rice seedlings were treated by plant activator protein. The expression of defense-related genes and resistance to TMV in transgenic plants were also detected. On one hand, we found that the expression of the 2 genes was similar in rice cultivars of Yuanfenzao and 1312 which respectively represents the resistant and sensitive cultivar although OsAiDG3 showed relatively high transcriptional level in two cultivars than OsAiDG4;however a dramatic expression change occurs in 1312 result from treatment of plant activator protein whereas relatively stable expression presents in Yuanfengzao. The result seemed to indicate that differences in gene expression of protein kinase in response to plant activator protein in different cultivars partly determined differences in blast resistance between different rice genotype. On the other hand, we selected plant expression vector PBI121 and used Agrvbacterium-medi&ted leaf disc transformation to get transgenic tobacco, which respectively overexpressed OsAiDG3 and OsAiDG4 gene. PCR and Northern blotting analysis revealed that successful transformation was achieved and the transformed genes were expressed in transgenic plants. Moreover, the elevated level of defense-related chitinase gene expression was found by Northern blotting analysis in transgenic tobacco although the basic expression level was low in nontransgenic tobacco. Lesions per leaf of TMV in OsAiDG3-o\erc\pressing and <2&4ï¿¡DG34-overexpressing plants was 64.1% and 52.5 compared with those of in nontransgenic plants. The present result seemed to suggest that the OsAiDG3 and OsAiDG4 may be involved in regulation of disease resistance defense.