Fangfang Li

Suppression of RNA silencing by a plant DNA virus satellite requires a host calmodulin-like protein to repress RDR6 expression.

In plants, RNA silencing plays a key role in antiviral defense. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that target different effector molecules in the RNA silencing pathway. Evidence has shown that plants also encode endogenous suppressors of RNA silencing (ESRs) that function in proper regulation of RNA silencing. The possibility that these cellular proteins can be subverted by viruses to thwart host defense is intriguing but has not been fully explored. Here we report that the Nicotiana benthamiana calmodulin-like protein Nbrgs-CaM is required for the functions of the VSR βC1, the sole protein encoded by the DNA satellite associated with the geminivirus Tomato yellow leaf curl China virus (TYLCCNV). Nbrgs-CaM expression is up-regulated by the βC1. Transgenic plants over-expressing Nbrgs-CaM displayed developmental abnormities reminiscent of βC1-associated morphological alterations. Nbrgs-CaM suppressed RNA silencing in an Agrobacterium infiltration assay and, when over-expressed, blocked TYLCCNV-induced gene silencing. Genetic evidence showed that Nbrgs-CaM mediated the βC1 functions in silencing suppression and symptom modulation, and was required for efficient virus infection. Moreover, the tobacco and tomato orthologs of Nbrgs-CaM also possessed ESR activity, and were induced by betasatellite to promote virus infection in these Solanaceae hosts. We further demonstrated that βC1-induced Nbrgs-CaM suppressed the production of secondary siRNAs, likely through repressing RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) expression. RDR6-deficient N. benthamiana plants were defective in antiviral response and were hypersensitive to TYLCCNV infection. More significantly, TYLCCNV could overcome host range restrictions to infect Arabidopsis thaliana when the plants carried a RDR6 mutation. These findings demonstrate a distinct mechanism of VSR for suppressing PTGS through usurpation of a host ESR, and highlight an essential role for RDR6 in RNA silencing defense response against geminivirus infection.

A versatile system for functional analysis of genes and microRNAs in cotton

Cotton is an important economic crop worldwide. Due to its long growth cycle, large genome size and recalcitrance to stable transformation, traditional methods for the analysis of gene function in this crop are difficult and labour intensive. Here, we report a cotton leaf crumple virus (CLCrV)-based vector and its application in gene function analysis through virus-induced gene silencing (VIGS) and overexpression of microRNAs (miRNAs), small tandem target mimic (STTM) and artificial miRNA (amiRNA) in cotton via an Agrobacterium-mediated infiltration approach. Using this system, we were able to efficiently silence two endogenous genes, magnesium chelatase subunit I (CHLI) and elongation factor-1α (EF-1α), in Gossypium species and the Bacillus thuringiensis cry1A gene in transgenic cotton. Furthermore, our results show that this vector can be used to ectopically express endogenous miR156 in G. hirsutum, causing a reduction in miR156-targeted RNA transcripts resulting in the development of abnormal leaf phenotypes. Ectopic expression of miR165/166 STTM with this vector led to downward curling and crumpled leaves, and a significant increase in the miR165/166 target mRNAs. This versatile system is easy to use and can provide more uniform and persistent gene silencing in cotton, thereby providing a powerful approach for gene discovery in cotton.

V2 of tomato yellow leaf curl virus can suppress methylation-mediated transcriptional gene silencing in plants.

Tomato yellow leaf curl virus (TYLCV) is a DNA virus belonging to the genus Begomovirus. TYLCV replicates using double-stranded DNA intermediates that can become the target of plant transcriptional gene silencing (TGS). Here, we show that the V2 protein of TYLCV can suppress TGS of a transcriptionally silenced green fluorescent protein (GFP) transgene in Nicotiana benthamiana line 16-TGS. Through bisulfite sequencing and chop-PCR, we demonstrated that the TYLCV V2 can reverse GFP transgene silencing by reducing the methylation levels in the 35S promoter sequence. Both AtSN1 and MEA-ISR loci in Arabidopsis thaliana were previously reported to be strongly methylated, and we show that the methylation status of both loci was significantly reduced in TYLCV V2 transgenic Arabidopsis plants. We conclude that TYLCV can efficiently suppress TGS when it infects plants, and its V2 protein is responsible for the TGS suppression activity.

Regulation of Nicotine Biosynthesis by an Endogenous Target Mimicry of MicroRNA in Tobacco

Endogenous target mimecry of an miRNA affects nicotine biosynthesis. The interaction between noncoding endogenous target mimicry (eTM) and its corresponding microRNA (miRNA) is a newly discovered regulatory mechanism and plays pivotal roles in various biological processes in plants. Tobacco (Nicotiana tabacum) is a model plant for studying secondary metabolite alkaloids, of which nicotine accounts for approximately 90%. In this work, we identified four unique tobacco-specific miRNAs that were predicted to target key genes of the nicotine biosynthesis and catabolism pathways and an eTM, novel tobacco miRNA (nta)-eTMX27, for nta-miRX27 that targets QUINOLINATE PHOSPHORIBOSYLTRANSFERASE2 (QPT2) encoding a quinolinate phosphoribosyltransferase. The expression level of nta-miRX27 was significantly down-regulated, while that of QPT2 and nta-eTMX27 was significantly up-regulated after topping, and consequently, nicotine content increased in the topping-treated plants. The topping-induced down-regulation of nta-miRX27 and up-regulation of QPT2 were only observed in plants with a functional nta-eTMX27 but not in transgenic plants containing an RNA interference construct targeting nta-eTMX27. Our results demonstrated that enhanced nicotine biosynthesis in the topping-treated tobacco plants is achieved by nta-eTMX27-mediated inhibition of the expression and functions of nta-miRX27. To our knowledge, this is the first report about regulation of secondary metabolite biosynthesis by an miRNA-eTM regulatory module in plants.

NbPHAN, a MYB transcriptional factor, regulates leaf development and affects drought tolerance in Nicotiana benthamiana

MYB transcriptional factors, characterized by the presence of conserved DNA-binding domains (BDs) (MYB domain), are involved in diverse processes including plant growth, development, metabolic and stress responses. In this study, a new R2R3-type MYB gene, NbPHAN (Nicotiana benthamiana PHANTASTICA), was identified in N. benthamiana. The NbPHAN encodes a protein of 362 amino acids and shares high sequence identities with the AS1-RS2-PHANs (ARPs) from other plant species. The NbPHAN protein targets to and forms homodimers in the nucleus. The MYB domain and C-terminal region of NbPHAN determine its subcellular localization and homodimerization, respectively. Using virus-induced gene silencing, we showed that the NbPHAN-silenced leaves exhibited severe downward curling and abnormal growth of blades along the main veins through suppressing the expression of the NTH20 gene. In addition, we found NbPHAN plays an important role in drought tolerance. The NbPHAN-silenced plants exhibited severe wilting and increased rate of water loss than that found in the non-silenced plants when growing under the water deficit condition. Although abscisic acid accumulation was not altered in the NbPHAN-silenced plants as compared with that in the non-silenced plants, several other stress-inducible genes were clearly repressed under the water deficit condition. Our results provide strong evidence that other than controlling leaf development, the ARP genes can also regulate plant tolerance to drought stress.

A calmodulin-like protein suppresses RNA silencing and promotes geminivirus infection by degrading SGS3 via the autophagy pathway in Nicotiana benthamiana

A recently characterized calmodulin-like protein is an endogenous RNA silencing suppressor that suppresses sense-RNA induced post-transcriptional gene silencing (S-PTGS) and enhances virus infection, but the mechanism underlying calmodulin-like protein-mediated S-PTGS suppression is obscure. Here, we show that a calmodulin-like protein from Nicotiana benthamiana (NbCaM) interacts with Suppressor of Gene Silencing 3 (NbSGS3). Deletion analyses showed that domains essential for the interaction between NbSGS3 and NbCaM are also required for the subcellular localization of NbSGS3 and NbCaM suppressor activity. Overexpression of NbCaM reduced the number of NbSGS3-associated granules by degrading NbSGS3 protein accumulation in the cytoplasm. This NbCaM-mediated NbSGS3 degradation was sensitive to the autophagy inhibitors 3-methyladenine and E64d, and was compromised when key autophagy genes of the phosphatidylinositol 3-kinase (PI3K) complex were knocked down. Meanwhile, silencing of key autophagy genes within the PI3K complex inhibited geminivirus infection. Taken together these data suggest that NbCaM acts as a suppressor of RNA silencing by degrading NbSGS3 through the autophagy pathway.

Artificial TALE as a Convenient Protein Platform for Engineering Broad-Spectrum Resistance to Begomoviruses

Transcription activator–like effectors (TALEs) are a class of sequence-specific DNA-binding proteins that utilize a simple and predictable modality to recognize target DNA. This unique characteristic allows for the rapid assembly of artificial TALEs, with high DNA binding specificity, to any target DNA sequences for the creation of customizable sequence-specific nucleases used in genome engineering. Here, we report the use of an artificial TALE protein as a convenient platform for designing broad-spectrum resistance to begomoviruses, one of the most destructive plant virus groups, which cause tremendous losses worldwide. We showed that artificial TALEs, which were assembled based on conserved sequence motifs within begomovirus genomes, could confer partial resistance in transgenic Nicotiana benthamiana to all three begomoviruses tested. Furthermore, the resistance was maintained even in the presence of their betasatellite. These results shed new light on the development of broad-spectrum resistance against DNA viruses, such as begomoviruses.

Development and application of an efficient virus-induced gene silencing system in Nicotiana tabacum using geminivirus alphasatellite

Virus-induced gene silencing (VIGS) is a recently developed technique for characterizing the function of plant genes by gene transcript suppression and is increasingly used to generate transient loss-of-function assays. Here we report that the 2mDNA1, a geminivirus satellite vector, can induce efficient gene silencing in Nicotiana tabacum with Tobacco curly shoot virus. We have successfully silenced the β-glucuronidase (GUS) gene in GUS transgenic N. tabacum plants and the sulphur desaturase (Su) gene in five different N. tabacum cultivars. These pronounced and severe silencing phenotypes are persistent and ubiquitous. Once initiated in seedlings, the silencing phenotype lasted for the entire life span of the plants and silencing could be induced in a variety of tissues and organs including leaf, shoot, stem, root, and flower, and achieved at any growth stage. This system works well between 18–32 °C. We also silenced the NtEDS1 gene and demonstrated that NtEDS1 is essential for N gene mediated resistance against Tobacco mosaic virus in N. tabacum. The above results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in N. tabacum.

Two Lysin-Motif Receptor Kinases, Gh-LYK1 and Gh-LYK2, Contribute to Resistance against Verticillium wilt in Upland Cotton

Lysin-motif (LysM) receptor kinases (LYKs) play essential roles in recognition of chitin and activation of defense responses against pathogenic fungi in the model plants Arabidopsis and rice. The function of LYKs in non-model plants, however, remains elusive. In the present work, we found that the transcription of two LYK-encoding genes from cotton, Gh-LYK1 and Gh-LYK2, was induced after Verticillium dahliae infection. Virus-induced gene silencing (VIGS) of Gh-LYK1 and Gh-LYK2 in cotton plants compromises resistance to V. dahliae. As putative pattern recognition receptors (PRRs), both Gh-LYK1 and Gh-LYK2 are membrane-localized, and all three LysM domains of Gh-LYK1 and Gh-LYK2 are required for their chitin-binding ability. However, since Gh-LYK2, but not Gh-LYK1, is a pseudo-kinase and, on the other hand, the ectodomain (ED) of Gh-LYK2 can induce reactive oxygen species (ROS) burst in planta, Gh-LYK2 and Gh-LYK1 may contribute differently to cotton defense. Taken together, our results establish that both Gh-LYK1 and Gh-LYK12 are required for defense against V. dahliae in cotton, possibly through different mechanisms.

SGS3 Cooperates with RDR6 in Triggering Geminivirus-Induced Gene Silencing and in Suppressing Geminivirus Infection in Nicotiana Benthamiana

RNA silencing has an important role in defending against virus infection in plants. Plants with the deficiency of RNA silencing components often show enhanced susceptibility to viral infections. RNA-dependent RNA polymerase (RDRs) mediated-antiviral defense has a pivotal role in resistance to many plant viruses. In RDR6-mediated defense against viral infection, a plant-specific RNA binding protein, Suppressor of Gene Silencing 3 (SGS3), was also found to fight against some viruses in Arabidopsis. In this study, we showed that SGS3 from Nicotiana benthamiana (NbSGS3) is required for sense-RNA induced post-transcriptional gene silencing (S-PTGS) and initiating sense-RNA-triggered systemic silencing. Further, the deficiency of NbSGS3 inhibited geminivirus-induced endogenous gene silencing (GIEGS) and promoted geminivirus infection. During TRV-mediated NbSGS3 or N. benthamiana RDR6 (NbRDR6) silencing process, we found that their expression can be effectively fine-tuned. Plants with the knock-down of both NbSGS3 and NbRDR6 almost totally blocked GIEGS, and were more susceptible to geminivirus infection. These data suggest that NbSGS3 cooperates with NbRDR6 against GIEGS and geminivirus infection in N. benthamiana, which provides valuable information for breeding geminivirus-resistant plants.