| Rice false smut caused by Ustilaginoidea virens is an important disease that effect rice production.The toxin produced by U.virens is also a great threat to food security.In this study,the differences in the infection of U.virens on resistant and susceptible varieties were observed at the histological level.The invasive period and the infection process of U.virens were studied.A highly efficient T-DNA insert mutant library of U.virens was constructed using the ATMT method.Sporulation-associated mutants were screened and functional analysis of the sporulation-related gene UvPRO1 was performed.The main results of the study are as follows:1.Resistant variety IR28 and susceptible variety Wanxian 98 were inoculated by conidia of GFP-labeled U.virens at the booting stage(7-8th differentiation stage of young panicle)and the infection process of U.virens on two varieties of panicles was observed under the stereo fluorescence microscope and confocal laser scanning microscope.The results showed that the germination rates of conidia on the surface of glumes on resistant and susceptible cultivars were 11.4%,38.1%,42.5%,56.7%,57.2%and 12.7%,36.5%,46.3%,73.4%,75.7%at 6,12,24,48 and 72 hours post inoculation(hpi),respectively.The germination rate of U.virens on resistant cultivar was significantly lower than that of susceptible cultivar at 48-72 hpi.At 3-15 days post inoculation(dpi)with susceptible cultivar,hyphae grew on the outer surface of the glumes and extended from outer to inner surfaces of the glumes through cracks between palea and lemma.Hyphae colonized on the surface of stamen and pistil,and infected filaments,anther,style and stigma,gradually.Then,floral organ were surrounded by a large number of mycelia and large mycelial masses grew out of the spikelets with yellow smut balls formed.In the resistant varieties,the infection process of U.virens was basically the same as the susceptible variety,but the proportion of colonization on inner surface of glumes of U.virens on resistant cultivar decreased by 86.9%-92.3%than susceptible cultivar,and the time of infecting style and stigma on resistant cultivars was 3 days longer than that of susceptible cultivars,while the infection rate of style and stigma decreased by 92.2%-95.3%and 92.5%-95.7%.At 15 dpi,the disease incidences of resistant and susceptible varieties were 7.5%and 89.1%,respectively,and the number of smut balls per panicle were 4.6 and 31.7,respectively.The above results showed that the germination of conidia on the surface of glumes and the infection process of mycelia from the outer surface of the glumes into the inner surface of the glumes were inhibited on resistant cultivar,and infection time was delayed and infection rate of flower was decreased,thus missing the chance of infect spikelets before rice pollination,and led to a significant reduction in the incidence of diseased.2.Rice plants at bud stage,tillering stage,booting stage and flowering stage were respectively inoculated by conidia of GFP-labeled U.virens,and the infection process after inoculation was observed,and GFP primers were used to detect U.virens from different tissues of different growth stages after inoculation.The results showed that:(1)At 1-3 days after the inoculation at budding stage,conidia adhered to the surface of radicle and root hairs and formed hyphae,and hyphae invaded the intercellular space of radicle through the base of the root hair and lateral root.A large number of hyphae grew on lateral roots,and extended to coleoptiles at 5 dpi.Hyphae extended from coleoptiles to incomplete leaf at 7 dpi.Green fluorescent hyphae was observed on the surface of leaf sheaths of very few rice plants at 30 dpi(tillering stage)and 90 dpi(booting stage).The presence of U.virens was detected by PCR in each part of plants at different stage after inoculation.(2)At 1-7 days after the inoculation at tillering stage,conidia adhering to the surface of leaf sheaths and stems germinated and formed hyphae,and extended on the surface of leaf sheaths.However,no hyphae was detected by PCR in each part of plant at different stage after inoculation.(3)At 1-3 days after spraying inoculation at booting stage,hyphae grew on the surface of the leaf sheath.At 5-12 dpi,U.virens was not observed on the surface of the glumes,stamen and pistil,but the pathogen was detected by PCR in panicle and leaf at the flowering stage and dough stage.However,at 5-12 days after injection inoculation at the booting stage,hyphae extended from outer to inner surface of the glumes through cracks between palea and lemma,and infected filaments,anther,style and stigma,and finally formed smut balls with 93.3%of incidence rate and31.4 smut balls per panicle,and pathogen was detected in each part of plant at flowering stage and dough stage.The relative expression levels of OsPromln2,OsRISBZ1 and OsGlutln1 genes associated with grain-filling in spikelets were detected by real-time PCR at 15 dpi.The expression levels of them in infected spikelets with smut ball formed was13.2 fold,43.5 fold and 28.6 fold higher than that of infected spikelets without smut balls formed,that indicated the activation expression of grain-filling related genes is a key factor in formation of smut balls.(4)At 1-12 days after spraying inoculation at flowering stage,hyphae grew on the surface of glumes and expanded to the inner surface of glumes and the base of ovary,but no hyphae were observed infected filaments,anther,style,stigma and ovary,and pathogen was detected existence in panicle at dough stage by PCR.The above results showed that the conidia of U.virens could germinate and produce hyphae on the surface of roots,stems,leaf and spikelets,and spread distance of the mycelia was observed to be inconsistent with different inoculation methods.Except for inoculation at tillering stage,pathogen was detected in plants after inoculation at different stage by PCR.After inoculation at budding stage,hyphae infected radicle and extended to the coleoptiles,incomplete leaves,and leaf sheath.After injection inoculation at booting stage,direct evidence from initial infection occurrence to smut ball formed on rice spikelets was observed,indicating that the formation of smut balls is closely related to the ability of infection style and stigma by pathogen before spikelets fertilized.Once spikelets have been fertilized,even if the pathogen spread to the inside of spikelets,hyphae can not obtain the nutrients and form rice balls.Thus,U.virens can not cause the disease on spikelets at flowering stage.3.A highly efficient Agrobacterium-mediated transformation(ATMT)system of U.virens was established.In transformation process,the concentration of conidia were 10~5spores/m L,the co-cultivation temperature was 24?,the co-culture time was 4 days,the AS concentration was 200?mol/L and the hygromycin concentration in selection of transformants was 200?g/m L,and transformation efficiency is 232.5 transformants per10~5 spores.3016 transformants with stable hygromycin resistance were obtained through ATMT system.Sporulation-associated mutants T-133,T-420,T-896,T-1296,and T2328 were screened,and Inverse-PCR and Tail-PCR method were used to analysis of the T-DNA flanking sequences.The result revealed that the T-DNA insertion disrupted genes of the mutants T-420 and T-1296 had lower homology with putative proteins in fungi,which function has not been reported.The gene disrupted by the T-DNA insertion of the mutant T-133 is homologous to the transcriptional regulatory protein PRO1 in Metarhizium acridum.The gene disrupted by the T-DNA insertion of the mutant T-896 is homologous to the serine/threonine protein phosphatase in Metarhizium acridum.The gene disrupted by the T-DNA insertion of the mutant T-2328 is homologous to the polysaccharide synthase Cps1 in Fusarium fujikuroi.4.The function of the sporulation-related gene UvPRO1 of the T-133 mutant was analyzed.The results showed that the growth rate of the knockout transformant?UvPRO1-27 was 2.20 mm/d on PSA plates for 6 days,which was significantly lower than complementary transformant C?UvPRO1-27(2.76 mm/d)and the wild-type strain(2.73 mm/d);in the PS liquid medium,the wild-type strain and the complementary transformant produced a large number of conidia after 7 days,and the sporulation of conidia were 6.72×10~6/m L and 6.82×10~6/m L,respectively,but conidia was not observed in the knockout transformant?UvPRO1-27.Different concentration of NaCl(0.1-0.5mol/L),SDS(0.01-0.05%)and CR(30-70?g/m L)were used to determine the effect of salt stress pressure,cell membrane and cell wall pressure on the knockout transformants,and the results showed that the knockout transformant?UvPRO1-27 showed higher sensitivity than wild-type strains and complemented transformants under each stress conditions.At 1-3 days after inoculation at the booting stage(7-8th differentiation stage of young panicle),mycelia of knockout transformant?UvPRO1-27 grew and extended on the outer surface of the glumes.After 4 days of inoculation,mycelial growth of?UvPRO1-27 on the outer surface of the glumes was inhibited,and until 12 days after inoculation,no hyphae were observed extending to the inside of the spikelet.No hyphae of?UvPRO1-27 was observed on the inner surface of the glumes,stamen and pistil.In contrast,mycelia of the wild-type strain and complementary transformant could spread from the outer surface of the glumes to the inner surface of the glumes,and infect the spikelet and form smut ball on spikelet.The above results indicated that UvPRO1 gene has a significant regulatory effect on mycelial growth,sporulation,stress resistance and pathogenicity of U.virens. |
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