| Sugarcane streak mosaic virus(SCSMV;Poacevirus geus of Potyviridae family)which is one of the pathogen causing sugarcane mosaic disease mainly occurred in some Asian countries including China.This study designed a pair primers and a TaqMan probe targeted at SCSMV CP sequence,then established a protocol of a one-step real-time quantitative RT-PCR(qRT-PCR)for SCSMV detection.The protocol had high specificity and no cross-reaction with the other sugarcane viruses such as SCMV,SrMV and SCYLV.Besides,the detection limit of the assay was 100 copies of ssRNA derived from transcription in vitro and 2 pg of total RNA extracted from SCSMV-infected leaves.A total of 35 field leaf samples with mosaic were diagnosed by qRT-PCR and conventional RT-PCR.Higher incidence of SCSMV infection was observed by qRT-PCR(68.6%)than the conventional RT-PCR assay(48.6%).The qRT-PCR assay was developed with good specificity,high sensitivity,and simpler and faster.This assay can be applied in SCSMV quarantine inspection and quarantine and disease epidemiological investigation.Besides,the complete genomic sequences of two Chinese isolates(YN-YZ2I1 and HN-YZ49)and a Burmese isolate(MYA-Formosa)were cloned and sequenced.The three isolates shared 96.8-99.5%of genomic nucleotide sequences identity(nt)and 98.9-99.6%of amino acid(aa)sequences identity with each other,and were 81.5-98.8%(nt)and 94.0-99.6%(aa)with other geographic isolates.Phylogenetic tree based on aa sequences of polyprotein revealed ten SCSMV isolates were clustered into two distinct phylogroups(G-1 and G-2).G-1 clade included eight isolates come from China,Buram,Japan,India,Indonesia and Thailand,and G-2 clade contained Indian isolate(IND671)and Pakistani isolate(PAK).High genetic variation between G-1 and G-2 groups was observed with 80.6-81.8%and 94.0-95.3%identifies of the genome nt sequences and amino acid sequence of polyprotein,respectively.Presence of obvious genetic differentiation between groups was supported by less frequent gene flow.However,within G-1 group was no obvious genetic differentiation and had frequent gene flow and homologous recombination.Selection pressure analysis suggested a positive amino acid residue sites in P1 protein.Finally,P1 protein encoded by SCSMV with strong RNA silencing suppressor was identified by transgenic transient expression in sugarcane young leaf segments and Nicotiana benthamiana 16 line via gene gun bombardment and Agrobacterium-mediated coinfiltration,respectively.The SCSMV P1,HC-Pro and P1/HC-Pro were introduced into sugarcane young leaf segments cells along with the EYFP reporter gene.The results showed that PI protein gradually increased the expression level of exogenous EYFP gene.At 72 h post-bombardment,EYFP expression cell counts and fluorescence intensity were remarkable increased by 1.74-fold and 2.27 folds,respectively,compared with the negative control(pUbi-Nos).P1/HC-Pro protein slightly enhanced EFYP fluorescence expression level,but not for HC-Pro protein.P1 significantly increased GFP fluorescence expression N.benthamiana 16 line leaves via Agro-infiltration.The mRNA expressive of PI and GFP reached the highest level at 3 dpi and were increased by 382 and 16 folds contracted to the negative control(pGD+pCHF3-GFP),respectively.PI/HC-Pro also enhanced a litter the GFP expression and the highest mRNA expression of PI/HC-Pro and GFP were found at 1 dpi.HC-Pro had no ability to improve the GFP expression.The P1 protein was proposed as a strong RNA silencing suppressor.In conclusion,we developed a protocol of efficiency,sensitive and rapid real-time qRT-PCR for SCSMV detection in this study.Then the molecular mechanism of SCSMV population genetic evolution and identification of P1 protein with RNA silencing suppressor were investigated.The findings provided a vital technical supporting for ecological controls of sugarcane mosaic disease and laid a foundation for further exploring molecular mechanisms of SCSMV interaction with plants. |
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