| Rice is one of the most important food crops in China.Rice false smut caused by Ustilaginoidea virens is one of the most destructive diseases in rice.U.virens can affect yield by reducing the panicle setting rate and produces toxin to threaten human and animal health.U.virens secrete effectors to promote the infection and location.Recently,many effector have been found to play an important role in pathogenicity of U.virens.The study on the effectors of U.virens is helpful to reveal its pathogenic mechanism and provide theoretical and research basis for the prevention and control of the disease.In this study,we investigate the function of UvScd1,an effector of U.virens,and evaluate its potentiality for disease control.The results are as follows:Functional analysis of UvScd1 protein in U.virens.In the early stages of this research,UvScd1 knockout,overexpressed and complemented transformants were obtained.The phenotypes of U.virens knockout and overexpressed transformants were observed.The growth rates of the knockout and overexpressed strains were decreased.Knockout and overexpression of UvScd1 decreased and increased conidiation,respectively.Knockout and overexpression of UvScd1 enhanced the tolerance cell membrane and cell wall stress.UvScd1 knockout increasetolerance to osmotic and oxidative stress,while overexpression decreased the tolerance.In addition,the pathogenicity of UvScd1 knockout mutants was reduced.The results showed that UvScd1 was involved in the growth,conidiation,and response to different stresses and contributed to the pathogenicity of U.virens.Analysis of key functional sites of UvScd1 protein.Previous studies showed that UvScd1 can interact with itself and inhibit dell death,and interact with rice expansin Os EXP.Sequence analysis showed that only two amino acids(75 Arg and 79 Phe)were different between UvScd1 and its homologous protein Mohrip2 in Magnaporthe oryzae,and 144 Asn of UvScd1 was the predicted glycosylation site.Three sites(75 Arg,79 Phe and 144 Asn)of UvScd1 protein were mutated,and their functions were analyzed.UvScd1 could not inhibit the cell death induced by XEG1,INF1 and BAX when 75 Arg and 79 Phe were mutated.The mutation of 144 Asn did not affect its function of inhibiting cell death.Mutations at the three sites affected the interaction with wild-type UvScd1 and Os EXP.The protein activity analysis showed that Os EXP had expansin activity,UvScd1 inhibited its activity.The ability to inhibit activity was reduced when three sites were mutanted.The results showed that UvScd1 could interact with Os EXP and inhibit its expansin activity.The 75 Arg,79 Phe and 144 Asn sites played important roles in dimer formation,cell death and Os EXP expansin inhibition.Analysis of ectopic expression of UvScd1 and Os EXP knockout rice.Compared with wild-type rice ZH11,rice expressing UvScd1 and knockout Os EXP had reduced resistance to U.virens,and the expression level of resistant-related genes in rice panicles was also reduced.Further analysis showed that the sucrose content in apoplast and the expression level of grain-filling-related genes increased in UvScd1 expressed and Os EXP knockout rice panicle.The results showed that UvScd1 and Os EXP were involved in regulating rice panicle resistance,extracellular sucrose content,and grain-filling-related gene expression.Study on UvScd1 protein for rice blast control.Previous studies have shown that rice leaves expressed UvScd1 exhibited lesion mimic symptoms,indicating that UvScd1 can trigger the resistance response in rice leaves.By expressing the UvScd1 protein in vitro and treating rice leaves,it was found that the UvScd1 protein could induce active oxygen burst of rice leaves,cause up-regulated expression of resistant-related genes,and enhance the resistance to M.oryzae.Basis on this,the Bacillus subtilis containing the UvScd1 is developed,the rice leaves treated by the B.subtilis exhibited active oxygen burst,the resistant-related genes were up-regulated,and the resistance to the rice blast was enhanced.The results showed that UvScd1 and B.subtilis containing UvScd1 could trigger the resistance response of rice leaves,which provided a basis for the development and field application of biocontrol engineered bacteria containing elicitor protein.In conclusion,the effector UvScd1 is involved in regulating the pathogenicity of U.virens.UvScd1 interacts with rice Os EXP and inhibits its expansin activity.UvScd1 can trigger the disease resistance response of rice leaves and was used to develop biocontrol engineered bacteria against rice blast.The results revealed the function and pathogenic mechanism of UvScd1 and provided a basis for developing biocontrol engineering bacteria and disease control. |
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