Molecular and physiological studies on oxidative burst and active defense mechanism in plant and its application for disease tolerance

本项目对马铃薯侵染染芽孢霉后氧化爆发（OXB）和主动防御网络的分子机制进行了初步研究。1)在防御反应过程中，依赖超氧化物生成NADPH氧化酶、RbohA和RbohB的立即反应型OXB和诱导型OXB被诱导，在诱导hr型防御反应中发挥关键的信号作用。2)RbohA是通过Ca^<2+>内流和Ca^<2+>依赖蛋白激酶激活的，而RbohB是在MAPK级联控制下诱导的，然后由CDPK激活，与第1 OXB相关。3) MAPKK的MEK1^<DD>的瞬时表达，经氨基酸交换修饰后具有组成活性，可激活MAPK的SIPK和WIPK，诱导RbohB、HMGR、PVS等，进而诱导hr型防御反应。4)激活的MPK1磷酸化多个宿主蛋白（PPS1-8），其中PPS3调节RbohB、HMGR和PVS的表达。5)亲和小种还诱导MAPK级联激活和非活性RbohB、HMGR和PVS的诱导，并刺激PVS3启动子连接GUS基因的转基因马铃薯中感染特异性GUS的表达。6)NO生成伴随着第1 OXB，在hr型反应的诱导中起重要作用。7) NO的生成依赖于诱导硝酸还原酶（NR）和其他未知酶。8)局部OXB刺激了内部组织细胞中连续的Ca^<2+>内流，该信号在RbohB和PAL表达诱导全身获得性耐药（SAR）后刺激了表面组织的全身OXB。9) PVS3启动子连接MEK^<DD>的转基因马铃薯植株表现出对毒力强的病原菌和茄疫病菌具有hr型防御反应的耐受性。

This project made it clear the followings on molecular mechanisms of oxidative burst (OXB) and active defense-network in Phytophtora infestans-infected potato plants. 1)In the course of the defense response, immediately-responsive OXB and induced OXB, which were dependent on superoxide-generating NADPH oxidase, RbohA and RbohB, were induced and then played a key signal role in the induction of HR-type defense response. 2)RbohA was activated through Ca^<2+> influx and a Ca^<2+>-dependent protein kinase, whereas RbohB was induced under control of MAPK cascade, and then activated by CDPK in relation to the 1st OXB. 3)A transient expression of MEK1^<DD> of MAPKK, that was modified by amino acid exchange to be constitutively active, caused activation of SIPK and WIPK of MAPK, induction of RbohB, HMGR,PVS etc. and then induction of HR-type defense response. 4)Activated MPK1 phosphorylated multiple host proteins (PPS1-8),PPS3 of which regulated expressions of RbohB, HMGR, and PVS. 5)Compatible races also induced activation of MAPK cascade and induction of non-active RbohB,HMGR and PVS, and stimulate infection-specific GUS expression in transgenic potato with PVS3 promoter-linked GUS gene. 6)NO generation was found to be accompanied by the 1st OXB, playing an important role in induction of HR-type response. 7)The NO generation was dependent on inducible-nitrate reductase (NR) and other unknown enzyme. 8)Local OXB stimulated a sequential Ca^<2+> influx among inner tissue cells, and this signal came to stimulate systemic OXB in the surface tissue, following expression of RbohB and PAL for induction of systemic acquired resistance (SAR). 9)Transgenic potato plants with PVS3 promoter-linked MEK^<DD> demonstrated tolerance to virulent P.infestans and Alternaria solani with HR-type defense response.