Development Of Conidiation Media For Ustilaginoidea Virens And Study On The Function Of Three Effectors UvScd1,UvTcd1 And UvNLP

Rice false smut disease caused by Ustilaginoidea virens is a panicle disease,and has become a serious production constraint in almost all rice-growing areas worldwide.In this study,we developed rice tissue media to stimulate conidiation of U.virens,which also are workable for the conidiation-defective isolates.Meanwhile,three effectors,UvScd1,UvTcd1 and UvNLP,were studied from a large number of effectors secreted by U.virens in the process of infection.The main results are as follows:1.Compared with PSB medium,tissue media with rice leaves or panicles induced more conidia at individual time points.Among the tested media,0.10 g/mL panicle medium was most efficient for conidiation.Whereas,some rice leaf media increased conidiation more effectively than panicle media except 0.10 g/mL panicle medium,and certain non-filtered tissue media were better than filtered counterparts.Although the conidia induced in rice tissue media were smaller,they were able to germinate on potato sucrose agar medium and infect rice normally.The rice tissue medium is also workable in inducing conidia for conidiation-defective isolates.This method provides a foundation for the production of conidia by U.virens,which avoids degradation of conidiation with transfers or long time storage in the laboratory and will be widely applicable in pathogenicity test as well as in genetic analyses for false smut resistance in rice cultivars.2.The effector UvScd1,highly expressed during the early stage of infection of U.virens in rice,could inhibit Bax or XEG1-induced hypersensitive cell death in Nicotiana benthamiana.The UvScd1 contains 6 cysteines and these cysteines were indispensable for the cell death suppression except 127 cysteine.The transient expression of UvScd1 in N.benthamiana leaves promoted the infection of Phytophthora capsica,and UvScd1-overexpression line OE-70 was more sensitive to U.virens.These results indicate that UvScd1 plays an important role in pathogenicity of U.virens.UvScd1 had an alkalization function that could increase the extracellular pH.Furthermore,UvScd1 interacted with rice expansion protein OsEXP.These results indicated that UvScd1 might promote the infection by adjusting the pH value in rice,and gave a novel understanding of the interaction mechanism between U.virens and rice.3.UvTcd1 only existed in U.virens and no homologous genes were found in other fungi.During the early stage of infection,the expression of UvTcd1 was up-regulated.UvTcd1 could trigger hypersensitive cell necrosis and induce expression of resistance related genes.Further study showed that some U.virens effectors were able to inhibit hypersensitive response induced by UvTcd1.These results indicate that plants could generate immune response triggered by UvTcd1,but effectors secreted by U.virens could suppress the plant immune response.Longer time was needed for UvTcd1-induced necrosis than R3a and Avr3a,or XEG1.It was proved by using virus-induced gene-silencing system that UvTcd1-induced necrosis was not mediated by BAK1 or SOBIR1 receptors.The candidate proteins interacted with UvTcd1 were screened using yeast two-hybrid.Altogether 18 candidate proteins were screened and 10 of them could interact with UvTcd1 which was verified by one-to-one manner.The ten candidate genes corresponding to the candidate protein are LOC_Os06g51150、LOC_Os05g41220、LOC_Os10g08550、LOC_Os02g06592、LOC_Os06g43800、LOC_Os03g08050、LOC_Os02g56850、LOC_Os04g45990、LOC_Os01g09860 and LOC_Os05g41172.4.A protein containing NLP-specific NPP1 domain was identified in U.virens and named UvNLP.UvNLP protein contained 302 amino acids,including eight cysteines and a functional signal peptide.Phylogenetic analysis revealed that UvNLP was a type 2 NLP.Real-time PCR revealed that UvNLP was highly expressed at 3 days post-inoculation.Furthermore,transient expression of UvNLP in N.benthamiana induced weak cell death,and replacement of the SP with that of Avr1b enhanced cell death.Real-time PCR showed that UvNLP induced the expression of ethylene-synthesis related and plant defence related genes.These results indicated that UvNLP was involved in infection during the early stage and provided evidence that NLPs acted as proteinaceous MAMPs.