Rho蛋白家族在丛枝菌根真菌与植物共生关系建立中的功能分析

Functional analysis of Rho protein family during the establishment of arbuscular mycorrhizal fungus-plant symbiosis

Rho家族蛋白是一类含有GTPase结构域的高度保守蛋白的总称,它是真核生物中Ras超家族主要成员之一。通过同源比对以及RACE等技术从丛枝菌根真菌(Rhizophagus irregularis)DAOM197198中分离到了3个Rho家族小G蛋白。通过与其他真菌Rho蛋白家族成员进行系统进化分析,结果表明,所分离到的Ri CDC42,RiRac1,RiRho1属于高度保守的Rho蛋白家族成员;利用实时荧光定量PCR方法对这3个基因在前共生阶段和共生阶段的表达模式进行分析,结果发现RiRho1在萌发孢子中的表达量最高,而在共生时期表达下调,RiRac1和Ri CDC42基因在共生时期的表达量比共生前期高。此外,本研究还利用酵母CDC42,Rho1温度敏感性突变株,稻瘟病菌ΔMg CDC42突变株以及HIGS技术对Rhizophagus irregularis Rho蛋白家族成员进行进一步研究。结果显示,RiRho1与Ri CDC42基因能够互补酵母Rho1与CDC42温度敏感型表型;对Ri CDC42与RiRac1进行HIGS时,Rhizophagus irregularis丛枝发生明显的降解;Ri CDC42基因能够互补稻瘟病菌ΔMg CDC42突变体部分表型,但不同的是互补菌株不产黑色素。由此推测Rho蛋白家族成员可能在丛枝菌根真菌共生关系建立的过程中具有重要的作用。

Rho protein family,a core member of the Ras superfamily in eukaryotes,contains the highly conserved GTPases domain. Three small Rho proteins were isolated from arbuscular mycorrhizal fungi Rhizophagus irregularis DAOM197198 through homolog research and RACE PCR strategies. Using real time q PCR method,the transcription profiles of the three genes during pre-symbiotic and symbiotic stages were analyzed. To further study the roles of RiRho1 and Ri CDC42 genes,complementary experiments were carried out in yeast Ts mutant and fungal pathogen Magnaporthe oryzae ΔMg CDC42. The results indicated that RiRho1,RiRac1 and Ri CDC42 proteins were highly similar to Rho proteins in M. oryzae. RiRho1 was highly induced in germination spores,whereas its transcripts were lower during the AM symbiosis. In contrast,expressions of RiRac1 and Ri CDC42 were significantly higher at symbiotic stages than that in pre-symbiotic stage. RiRho andRi CDC42 can partially rescue the yeast mutant. Furthermore,the Ri CDC42 was able to rescue the phenotype of the M.oryzae CDC42 mutant. However,the mutant expressing Ri CDC42 gene did not generate the melanin. The roles RiRac1 and Ri CDC42 played were further confirmed by host induced gene silence（ HIGS）. Taken as a whole,the above data and results suggested that the Rho protein may play important roles during the arbuscular mycorrhizal symbiosis.