Tobacco Rattle Virus-induced Phytoene Desaturase (PDS) Silencing in Centaurea cyanus

Virus-induced gene silencing(VIGS)is a genetic tool used to assess gene function.Tobacco rattle virus(TRV)is a VIGS vector commonly used to induce endogenous gene silencing in plants.However,there is no VIGS system established for Centaurea spp.We evaluated the effectiveness of a TRV-based VIGS system using phytoene desaturase(PDS)as a reporter gene in Centaurea cyanus.Three methods including pressure-,vacuum-and apical meristem-infiltrationwere tested to infect C.cyanus seedlings.Photobleached leaveswere only obtained using apicalmeristem-infiltration after a 14 d treatment.The CcPDS transcripts in photobleached leaves were significantly reduced compared with that in green leaves treated with empty TRV.Four C.cyanus cultivars were tested to detect their VIGS responses,and‘Dwarf Tom Pouce Blue’was the most sensitive.The agro-infiltration condition was optimized by screening for the optimal seedling stage as well as the optimum Agrobacterium density for efficient silencing.Seedlings with four true leaves and infiltration with an Agrobacterium density of OD_(600)0.5 were optimal conditions to obtain more photobleached leaves and more intense photobleached phenotype.The results demonstrated the feasibility of TRV-based VIGS for functional analysis of genes in C.cyanus.

Host-Induced Gene Silencing of Effector AGLIP1 Enhanced Resistance of Rice to Rhizoctonia solani AG1-IA

Rice sheath blight, caused by Rhizoctonia solani AG1-IA, is a major disease in rice-growing areas worldwide. Effectors of phytopathogenic fungi play important roles during the infection process of fungal pathogens onto their host plants. However, the molecular mechanisms by which R. solani effectors regulate rice immunity are not well understood. Through prediction, 78 candidate effector molecules were identified. Using the tobacco rattle virus-host induced gene silencing(TRV-HIGS) system, 45 RNAi constructs of effector genes were infiltrated into Nicotiana benthamiana leaves. The results revealed that eight of these constructs resulted in a significant reduction in necrosis caused by infection with the AG1-IA strain GD-118. Additionally, stable rice transformants carrying the double-stranded RNA construct for one of the effector genes, AGLIP1, were generated to further verify the function of this gene. The suppression of the AGLIP1 gene increased the resistance of both N. benthamiana and rice against GD-118, and also affected the growth rate of GD-118, indicating that AGLIP1 is a key pathogenic factor. Small RNA sequencing showed that the HIGS vectors were processed into si RNAs within the plants and then translocated to the fungi, leading to the silencing of the target genes. As a result, AGLIP1 might be an excellent candidate for HIGS, thereby enhancing crop resistance against the pathogen and contributing to the control of R. solani infection.

Virus-induced gene silencing(VIGS)in Chinese narcissus and its use in functional analysis of NtMYB3

Virus-induced gene silencing(VIGS)is a natural defense mechanism of plants,which can cause sequence-specific degradation of viral RNA and is often used for studying the functional genome.At present,the VIGS system mediated by Tobacco rattle virus(TRV)has been successfully established in many plant species.However,the VIGS system in Chinese narcissus had not been reported yet.In this paper,the first use of the TRV-VIGS system in Chinese narcissus is described in detail.By injecting pTRV2-GFP into narcissus leaves,we confirmed that TRV could infect narcissus and move in narcissus plants.After the inoculation of pTRV2-Nt PDS by two methods,phenomena such as photo-bleaching appeared in narcissus leaves and qRT-PCR results proved that PDS gene of narcissus was successfully silenced.Besides,pTRV2-NtMYB3 was inoculated into basal plates of narcissus to study the function of NtMYB3;the results showed that NtMYB3 is an inhibitor of flavonoids biosynthesis,which can increase the content of proanthocyanidins in basal plates of narcissus by repressing the expression level of NtFLS.These results indicate that the TRV-VIGS system has been successfully established in Chinese narcissus and successfully applied to the functional study of NtMYB3.

Virus-induced Gene Silencing in Eggplant(Solanum melongena)

Eggplant （Solanum melongena） is an economically important vegetable requiring investigation into its various genomic functions. The current limitation in the investigation of genomic function in eggplant is the lack of effective tools available for conducting functional assays. Virus-induced gene silencing （VIGS） has played a critical role in the functional genetic analyses. In this paper, TRV-mediated VIGS was successfully elicited in eggplant. We first cloned the CDS sequence of PDS （PHYTOENE DESATURASE） in eggplant and then silenced the PDS gene. Photo-bleaching was shown on the newly-developed leaves four weeks after agroinoculation, indicating that VIGS can be used to silence genes in eggplant. To further illustrate the reliability of VIGS in eggplant, we selected Chl H, Su and CLA1 as reporters to elicit VIGS using the high-pressure spray method. Suppression of Chl H and Su led to yellow leaves, while the depletion of CLA1 resulted in albino. In conclusion, four genes, PDS, Chl H, Su （Sulfur）, CLA1, were down-regulated significantly by VIGS, indicating that the VIGS system can be successfully applied in eggplant and is a reliable tool for the study of gene function.

Efficiency for Gene Silencing Induction in Nicotiana Species by a Viral Satellite DNA Vector

Virus-induced gene silencing （VIGS） is a useful technique for rapid plant gene function analysis. We recently reported a new VIGS vector modified from Tomato yellow leaf curl China virus （TYLCCNV） DNAβ （DNAm β）. In this study we compared in detail DNAmβ-induced gene silencing in four Nicotiana species including N. benthamiana, N. glutinosa, N. tabacum and N. paniculata. We found that DNAmβ-induced gene silencing in the four species was distinct in developing dynamics, tissue specificity, efficiency, and constancy in the plant life span. It was most efficient in N. benthamiana, where development of VIGS was most rapid, without tissue specificity and nearly 100% efficient. DNAmβ-induced gene silencing in N. glutinosa was also efficient despite being slightly less than in N. benthamiana. It initially occurred in veins, later was scattered to mesophyll, finally led to complete silencing in whole leaves. In both species, VIGS constantly expressed until the plants died. However, DNAmβ-mediated VIGS in the other two Nicotiana species, N. tabacum and N. paniculata, was significantly less efficient. It was strictly limited within the veins of the silenced leaves, and constantly occurred only over 3-4 weeks. The upper leaves that emerged later stopped showing the silencing phenotype. DNAmβ-induced gene silencing in N. benthamiana and N. glutinosa was not significantly influenced by the growth stage when the plants were agro-inoculated, and was not sensitive to high growth temperature up to 32℃. Our results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in some Nicotiana species.