Zhongxin Guo

Tomato yellow leaf curl virus (TYLCV) is a devastating viral pathogen threatening agricultural crops globally. In this study, we identified a novel TYLCV isolate (TYLCV-YN6244), which was associated with disease outbreaks in resistant tomato cultivars in Yuanmou County in Yunnan Province, China. We determined the complete genome of TYLCV-YN6244 and found it encoded six viral proteins characteristic of Geminivirus. We identified its V2 protein as a potent viral suppressor of RNA silencing (VSR), and generated an infectious clone of wildtype TYLCV-YN6244, or V2-defective TYLCV-YN6244 (TYLCV-YN6244-ΔV2) in which V2 was deleted. Both infectious clones were capable of systemically infecting tobacco and tomato. However, TYLCV-YN6244 but not TYLCV-YN6244-ΔV2 could cause disease symptoms in wildtype tobacco or tomato plants, and viral accumulation was drastically reduced in plants infected with TYLCV-YN6244-ΔV2 compared to TYLCV-YN6244 while the efficiency of virus-derived small interfering RNAs (vsiRNAs) biogenesis was conversely increased in plants infected with TYLCV-YN6244-ΔV2. Surprisingly, small RNA profiling indicated that the 21nt and 22nt rather than 24nt vsiRNAs were predominantly produced in tomato plants infected with either TYLCV-YN6244 or TYLCV-YN6244-ΔV2. It is likely that 21nt and 22nt vsiRNAs play a significant role in plant resistance to DNA viruses. Furthermore, transcriptome analyses revealed that TYLCV-YN6244 or TYLCV-YN6244-ΔV2 infection differentially modulated metabolism and defense-related pathways in tomato, probably underlying distinct viral pathogenicity and disease symptoms induced in plants. Overall, our research not only identified a novel pathogenic TYLCV isolate but also characterized molecular biology and host response in tomato with infectious clones developed in this study, with implications for untangling virus–host interaction for developing novel resistance in crop tomato.

Specific targeting of host genes by plant viruses leads to the induction of distinct disease symptoms with devastating consequences for agriculture. However, it is unknown whether plants have evolved genes to specifically inhibit the development of viral disease symptoms. Here we identified the causal gene in 2 Arabidopsis thaliana mutants that develop prominent systemic necrosis after infection with both wild-type cucumber mosaic virus (CMV) and its mutant (CMV-Δ2b), which is defective in the suppression of antiviral RNA interference. We found that antiviral RNAi 5 (AVI5) is located in a large chromosomal deletion in both Arabidopsis mutants. We demonstrate that AVI5 acts to inhibit CMV-Δ2b viral accumulation by promoting the biogenesis of virus-derived small interfering RNAs in the antiviral RNA interference pathway before the host amplifies these small interfering RNAs. We further show that cucumber mosaic virus infection induces systemic necrosis symptoms associated with cell death and a reactive oxygen species burst and that AVI5 potently inhibits them. Unlike its suppression of virus accumulation, however, AVI5 inhibits the development of systemic necrosis symptoms without reducing viral titers or inducing antiviral RNA interference. Together, our study demonstrates that Arabidopsis plants have evolved a dual functional gene for antiviral protection to inhibit viral infection and symptom development by distinct mechanisms.

A major threat to rice production is the disease epidemics caused by insect-borne viruses that emerge and re-emerge with undefined origins. It is well known that some human viruses have zoonotic origins from wild animals. However, it remains unknown whether native plants host uncharacterized endemic viruses with spillover potential to rice (Oryza sativa) as emerging pathogens. Here, we discovered rice tiller inhibition virus (RTIV), a novel RNA virus species, from colonies of Asian wild rice (O. rufipogon) in a genetic reserve by metagenomic sequencing. We identified the specific aphid vector that is able to transmit RTIV and found that RTIV would cause low-tillering disease in rice cultivar after transmission. We further demonstrated that an infectious molecular clone of RTIV initiated systemic infection and causes low-tillering disease in an elite rice variety after Agrobacterium-mediated inoculation or stable plant transformation, and RTIV can also be transmitted from transgenic rice plant through its aphid vector to cause disease. Finally, global transcriptome analysis indicated that RTIV may disturb defense and tillering pathway to cause low tillering disease in rice cultivar. Thus, our results show that new rice viral pathogens can emerge from native habitats, and RTIV, a rare aphid-transmitted rice viral pathogen from native wild rice, can threaten the production of rice cultivar after spillover.

Evolutionarily conserved antiviral RNA interference (RNAi) mediates a primary antiviral innate immunity preventing infection of broad-spectrum viruses in plants. However, the detailed mechanism in plants is still largely unknown, especially in important agricultural crops, including tomato. Varieties of pathogenic viruses evolve to possess viral suppressors of RNA silencing (VSRs) to suppress antiviral RNAi in the host. Due to the prevalence of VSRs, it is still unknown whether antiviral RNAi truly functions to prevent invasion by natural wild-type viruses in plants and animals. In this research, for the first time we applied CRISPR-Cas9 to generate ago2a, ago2b, or ago2ab mutants for two differentiated Solanum lycopersicum AGO2s, key effectors in antiviral RNAi. We found that AGO2a but not AGO2b was significantly induced to inhibit the propagation of not only VSR-deficient Cucumber mosaic virus (CMV) but also wild-type CMV-Fny in tomato; however, neither AGO2a nor AGO2b regulated disease induction after infection with either virus. Our findings firstly reveal a prominent role of AGO2a in antiviral RNAi innate immunity in tomato and demonstrate that antiviral RNAi evolves to defend against infection of natural wild-type CMV-Fny in tomato. However, AGO2a-mediated antiviral RNAi does not play major roles in promoting tolerance of tomato plants to CMV infection for maintaining health.

Evolutionarily conserved antiviral RNA interference (RNAi) mediates a primary antiviral innate immunity preventing the infection of broad spectrum viruses in plants. However, the detailed mechanism in plants is still largely unknown, especially in important agricultural crops including tomato. On the other aspect, varieties of pathogenic viruses evolve to possess Viral Suppressor of RNA silencing (VSR) to suppress antiviral RNAi in host. Due to the prevalence of VSR, it is still skeptical that antiviral RNAi truly functions to prevent the invasion of natural wildtype viruses in plants and animals. In the research, it is for the first time we applied CRISPR-Cas9 to generate ago2a, ago2b or ago2ab mutants for two differentiated Solanum lycopersicum AGO2, one key effector in antiviral RNAi. We found that AGO2a but not AGO2b was significantly induced to inhibit the propagation of not only VSR-deficient Cucumber mosaic virus (CMV) but also wildtype CMV-Fny in tomato, however, both AGO2a and AGO2b did not regulate disease induction after the infection of either virus. Our findings firstly reveal a prominent role of AGO2a in antiviral RNAi innate immunity in tomato and demonstrate that antiviral RNAi evolves to defend the infection of natural wildtype CMV-Fny in tomato, however AGO2a-mediated antiviral RNAi does not play major roles in promoting tolerance of tomato plants to CMV infection for maintaining health.

A major threat to rice production is the disease epidemics caused by insect-borne viruses that emerge and re-emerge with undefined origins. It is well known that some human viruses have zoonotic origins from wild animals. However, it remains unknown whether native plants host new endemic viruses with spillover potential to rice ( Oryza sativa ) as emerging pathogens. Here, we discovered rice tiller inhibition virus (RTIV), a novel RNA virus species, from colonies of Asian wild rice ( O. rufipogon ) in a genetic reserve by metagenomic sequencing. We identified the specific aphid vector to transmit RTIV and found RTIV would cause low-tillering disease in rice cultivar after transmission. We further demonstrated that an infectious molecular clone of RTIV initiated systemic infection and causes low-tillering disease in an elite rice variety after Agrobacterium-mediated inoculation or stable plant transformation, and RTIV can also be transmitted from transgenic rice plant through its aphid vector to cause disease. Finally, global transcriptome analysis indicated that RTIV may disturb defense and tillering pathway to cause low tillering disease in rice cultivar. Thus, our results show that new rice viral pathogens can emerge from native habitats, and RTIV, a rare aphid-transmitted rice viral pathogen from native wild rice, can threaten the production of rice cultivar after spillover.

Due to the impaired antiviral RNAi, the dcl2dcl4 (dcl2/4) mutant is highly susceptible to viruses deficient of the viral suppressor of the RNA silencing (VSR) contrast to wild-type Arabidopsis. It was found that more severe disease symptoms were induced in dcl2/4 infected with VSR-deficient CMV (CMV-Δ2b or CMV-2aTΔ2b) compared to wild-type Arabidopsis infected with intact CMV. In order to investigate the underlying mechanism, comparative transcriptome analysis was performed with Col-0 and dcl2/4 that were infected by CMV, CMV-Δ2b and CMV-2aTΔ2b, respectively. Our analysis showed that the systematic infection of CMV, CMV-Δ2b and CMV-2aTΔ2b could cause hypoxia response and reduce photosynthesis. Asymptomatic infections of CMV-Δ2b or CMV-2aTΔ2b in Columbia (Col-0) promoted the expression of cell division-related genes and suppressed the transcription of metabolism and acquired resistance genes. On the other hand, immunity and resistance genes were highly induced, but photosynthesis and polysaccharide metabolism-related genes were suppressed in diseased plants. More interestingly, cell wall reorganization was specifically caused in modestly diseased Col-0 infected by CMV and a strong activation of SA signaling were correspondingly induced in severely diseased dcl2/4 by CMV or CMV mutants. Thus, our research revealed the nature of the Arabidopsis–CMV interaction at the transcriptome level and could provide new clues in symptom development and antiviral defense in plants.

Multiple antiviral immunities were developed to defend against viral infection in hosts. RNA interference (RNAi)-based antiviral innate immunity is evolutionarily conserved in eukaryotes and plays a vital role against all types of viruses. During the arms race between the host and virus, many viruses evolve viral suppressors of RNA silencing (VSRs) to inhibit antiviral innate immunity. Here, we reviewed the mechanism at different stages in RNAi-based antiviral innate immunity in plants and the counteractions of various VSRs, mainly upon infection of RNA viruses in model plant Arabidopsis. Some critical challenges in the field were also proposed, and we think that further elucidating conserved antiviral innate immunity may convey a broad spectrum of antiviral strategies to prevent viral diseases in the future.

Geminivirus infection induces the expression of a plant imprinted E3 ligase gene, leading to the proteosomal degradation of DNA methyltransferases and epigeneti