禾谷镰孢Tri12基因敲除突变体的致病力

Significant reduction in pathogenicity in Fusarium graminearum caused by knock-out of Tri12,a trichothecene efflux pump gene

Tri12是镰孢菌Fusarium编码单端孢霉烯族毒素(trichothecenes,以下简称“单族毒素”)输出泵的基因,而产毒基因Tri5编码的单端孢霉二烯合酶催化毒素生物合成中的第一步反应。以禾谷镰孢FusariumgraminearumSchw.野生型菌株NRRL29169为亲本,获得Tri12敲除的转化子MT12。与NRRL29169相比,MT12在PDA培养基上生长慢,生长量小(P<0.01),大分生孢子较长。致病力测定结果显示,NRRL29169致病力最强,所致病害可以自接种点向上下扩展至其他小穗,而MT12的致病力极弱。我们由此推测,Tri12的敲除导致病菌产生的毒素不能被泵出体外毒害寄主,进而在病菌体内积累抑制自身生长和毒素的进一步产生,从而降低病菌的致病力。

Trichothecenes are secondary metabolites,some of which are pathogenicity factors in Fusarium graminearum.Tri12 is a gene encoding a trichothecene efflux pump that is responsible for the transportation of trichothecenes out of fungal cells.Part of the Tri12 coding region(1 011 bp)was PCR-amplified and cloned into the Sal Ⅰ/BamH Ⅰ sites of plasmid pCB1004.The resultant plasmid pCBTri12 was used to transform the wild type F.graminearum strain NRRL 29169 to generate a Tri12 knock-out mutant strain(MT12).On potato-dextrose-agar,MT12 grew slower and produced less mycelia than its parent strain NRRL 29169 (P<0.01),though macroconidia of MT12 were longer than those of NRRL 29169(P<0.05).Virulence on wheat was determined for NRRL 29169,MT12 and GZT40,a strain with the Tri5 gene disrupted.Tri5 encodes trichothecene synthase that catalyzes the first step of trichothecene biosynthesis.Among the three strains tested,NRRL 29169 was the most virulent,with disease symptoms spreading from the inoculated spikelets to other spikelets,while the two mutant strains were less virulent with no spreading being observed.From these experiments we speculate that disruption of Tri12 causes accumulation of trichothecenes in fungal mycelia instead of them being pumped into host tissues.We further hypothesize that trichothecenes are toxic to the fungus itself and that exposure to the toxins inhibits its growth and toxin production.As a result,the pathogenicity of the pathogen may then be greatly reduced.