| Potato virus X(PVX; genus Potexvirus) can infect a wide range of major crops in the family Solanaceae including potato, tomato, pepper and tobacco. PVX is often synergism with members of Potyvirus and resulting in more severe losses. Recently, Many studies about PVX genome structure and function, classification, mechanism of host resistance and virus control have been reported, however, few is known how to screen mild strain efficiently and what the pathogenesis mechanism is. In this study, attenuated PVX mutants using site-directed mutagenesis based on self-constructed infectious cDNA clone of PVX were obtained. Their cross protection efficiency against the wild strain were tested. Transcriptome sequencing data of Nicotiana benthamiana infected by wild type PVX(Pwt) and attenuated mutant(Pat) were compared. The potential application of modified attenuated PVX mutants as a tool to cross protects multiple viruses were tested using Tobacco mosaic virus(TMV)and Potato virus Y(PVY) as an example. The results are shown below:Firstly, we screened attenuated PVX mutants and tested their cross protection efficiency.Alanine replacement at postion Glu46, Asn863, Asn968 and Glu1001, respectively, in PVX RNA dependent RNA polymerase(RdRp), can significantly reduced PVX virulence in Nicotiana benthamiana plants. Western- and Northern blotting analysis showed that coat protein, plusand minus-sense RNAs accumulation level of these attenuated mutants was decreased significantly.The cross protection effeciency of these attenuated mutants was assayed on N.benthamiana. None attenuated mutants could provide cross protection against the wild type PVX infection with five days interval. E1001 A can provide 100% efficiency again Pwt with both 10 and 15 days interval. E46 A can delay the Pwt infection with 10 days interval and provide partial protection with 15 days interval, the protection efficiency was 33.3%. N863 A and N968 A can not provide cross protection against Pwt with either 10 or 15 days interval.Mutant dM that contained two mutations could only provided partial protection against Pwt infection with 15 days interval, the protection efficiency was 23.3%.Northern blotting analysis showed that the protection efficacy of the attenuated mutants against severe infection of PVX is positively related to the accumulation levels of siRNAproduced by attenuated mutants, suggesting that the mechanism of cross protection against PVX is RNA-mediated. However, the role of CP in cross protection can not be excluded.Then, we compared transcriptome data of N. benthamiana infected with Pwt and Pat.Digital gene expression profile sequencing technology(DGE) was used to analyze the transcriptome profiling of N. benthamiana infected with Pwt and Pat. At 1 dapi(days postagroinoculation), there are 467 significantly altered transcripts in Pwt-infected plants. GO analyses showed that differentially expressed genes(DEGs) caused by Pwt were primarily enriched in DNA metabolic process, DNA replication, chlorophyll biosynthetic process, chlorophyll metabolic process, pigment biosynthetic process, pigment metabolic process, magnesium chelatase activity categories. KEGG analyses showed DNA replication pathway was significantly enriched. At 2 dapi, there are 1160 significantly altered transcripts in Pwtinfected plant. GO analyses indicated that the DEGs caused by Pwt were enriched in photosynthesis, photosynthetic membrane, thylakoid, thylakoid part, photosystem I,photosystem II categories. KEGG analyses showed that photosynthesis-antenna protein,photosynthesis, carotenoid biosynthesis pathways were significantly enriched. At 10 dapi,Pwt-infected plants have 1693 significantly altered transcripts. GO analyses indicated that the DEGs were primarily enriched in the biological process category, within the biological process category, biological process, metabolic process, cellular process, organic substance metabolic process, primary metabolic process, cellular metabolic process, macromolecule metabolic process, cellular macromolecule metabolic process were significantly enriched.KEGG analyses showed that the significantly enriched pathway was ribosome. It is speculated that Pwt first used the host DNA replication and metabolic system to complete their own replication, and at the same time altered the gene expression related to photosynthetic pigments, which affected the photosynthesis. The change of photosynthesis affect a large number of signaling pathways such as biological process, metabolic process, cellular process,which may be the main reason for Pwt infection.Further analysis showed that the genes involved in most chlorophyll metabolism pathway, carotenoid biosynthesis pathway were up-regulated, the genes involved in most cellulose synthesis and cell-wall structure components were down-regulated may contribute to the severe symptoms induced by the Pwt. Besides, at the early stage of infection, Pwt mayinhibit salicylic acid and jasmonate signal transduction pathways to break the host resistance;in the late infection of Pwt, expression of genes involved in ubiqutin-proteasome pathway were changed suggested that PVX is likely to affect the ubiqutin-proteasome pathway to interfere with the host physiological reaction and cause disease.Lastly, we evaluated the potential of modifying attenuated PVX mutants as a tool to cross protect against multiple viruses. The fragments in TMV and PVY genome that can induce best cross protection efficiency against corresponding virus infection were screened.Corresponding fragments from TMV or PVY were inserted into E1001 A to examine their capacity to cross protect against TMV or PVY respectively. The results showed that the fragments RdRp-2 and PVY-7 from TMV and PVY can induce the best cross protection.RdRp-2 and PVY-7 were fused by overlap PCR and subsequently inserted to E1001 A. The new obtained construct can provide 33.3% and 44.4% cross protection efficiency to TMV and PVY, respectively. |
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