Apple geminivirus (AGV) is a recently identified geminivirus which is isolated from the apple tree in China. We carried out functional scanning of apple geminivirus proteins as symptom determinants and suppressors of posttranscriptional gene silencing (PTGS). Our results indicated that AGV V2 is an important virulence factor localized to the nucleus and cytoplasm that suppresses PTGS and induces severe symptoms of crinkling and necrosis. AGV C1 is also a virulence determinant which elicits systemic necrosis when expressed from a PVX-based vector. The AGV C4 is targeted to cytoplasm, plasma membrane, nucleus, and chloroplasts. The inoculation of PVX-C4 on N. benthamiana induced severe upward leaf curling, which implied that AGV C4 also functions as a symptom determinant, and mutation analyses suggested that the acylated residues on Gly2 and Cys8 play important roles in its subcellular localization and symptom development.

Paper mulberry (Broussonetia papyrifera) is a perennial woody plant used as source material for Cai Lun paper making, in traditional Chinese medicine, and as livestock feed. To identify the presence of viruses in paper mulberry plants affected by a disease with leaf curl symptoms, high-throughput sequencing of total RNA was performed. Analysis of transcriptome libraries allowed the reconstruction of two geminivirus-like genomes. Rolling-circle amplification and PCR with back-to-back primers confirmed the presence of two geminiviruses with monopartite genomes in these plants, with the names paper mulberry leaf curl virus 1 and 2 (PMLCV-1 and PMLCV-2) proposed. The genomes of PMLCV-1 (3,056 nt) and PMLCV-2 (3,757 to 3,763 nt) encode six proteins, with the V4 protein of PMLCV-1 and the V3 proteins of both viruses having low similarities to any known protein in databases. Alternative splicing of an intron, akin to that of mastre-, becurto-, capula-, and grabloviruses, was identified by small RNA (sRNA)-seq and RNA-seq reads mapping to PMLCV-1 and PMLCV-2 antisense transcripts. Phylogenetic analyses and pairwise comparisons showed that PMLCV-1 and PMLCV-2 are most closely related to, but distinct from, two unassigned geminiviruses, citrus chlorotic dwarf associated virus and mulberry mosaic dwarf associated virus, suggesting that they are two new members of the family Geminiviridae. Field investigation confirmed the close association of the two viruses with leaf curl symptoms in paper mulberry plants and that coinfection can aggravate the symptoms.

Maize chlorotic mottle virus (MCMV) infection causes growth abnormalities in maize. Transcriptome sequencing was conducted to compare the global gene expression of MCMV-inoculated plants with that of mock-inoculated plants. Data analyses showed that brassinosteroid (BR)-associated genes were upregulated after MCMV infection. Exogenous 2,4-epibrassinolide (BL) or brassinazole (BRZ) applications indicated that BR pathway was involved in the susceptibility to MCMV infection. In addition, treatment of BL on maize induced the accumulation of nitric oxide (NO), and the changes of NO content played positive roles in the disease incidence of MCMV. Moreover, MCMV infection was delayed when the BL-treated plants were applied with NO scavenger, which suggested that BR induced the susceptibility of maize to MCMV infection in a NO-dependent manner. Further investigation showed the maize plants with knock-down of DWARF4 (ZmDWF4, a key gene of BR synthesis) and nitrate reductase (ZmNR, a key gene of NO synthesis) by virus-induced gene silencing displayed higher resistance to MCMV than control plants. Taken together, our results suggest that BR pathway promotes the susceptibility of maize to MCMV in a NO-dependent manner.

Three RNA viruses—Cucumis melo cryptic virus (CmCV), Cucumis melo amalgavirus 1 (CmAV1), and melon necrotic spot virus (MNSV)—were identified from a melon (Cucumis melo) transcriptome dataset. CmCV has two dsRNA genome segments; dsRNA-1 is 1592 bp in size, containing a conserved RNA-dependent RNA polymerase (RdRp), and dsRNA-2 is 1715 bp in size, and encodes a coat protein (CP). The sequence alignment and phylogenetic analyses of the CmCV RdRp and CP indicated CmCV clusters with approved or putative deltapartitiviruses in well-supported monophyletic clade. The RdRp of CmCV shared an amino acid sequence identity of 60.7% with the closest RdRp of beet cryptic virus 3, and is <57% identical to other partitiviruses. CmAV1 is a nonsegmented dsRNA virus with a genome of 3424 bp, including two partially overlapping open reading frames (ORFs) encoding a putative CP and RdRp. The sequence alignment and phylogenetic analyses of CmAV1 RdRp revealed that it belongs to the genus Amalgavirus in the family Amalgaviridae. The RdRp of CmAV1 shares 57.7% of its amino acid sequence identity with the most closely related RdRp of Phalaenopsis equestris amalgavirus 1, and is <47% identical to the other reported amalgaviruses. These analyses suggest that CmCV and CmAV1 are novel species in the genera Amalgavirus and Deltapartitivirus, respectively. These findings enrich our understanding of new plant dsRNA virus species.

Tomato (Solanum lycopersicum) plants exhibiting severe leaf distortion, mottle and systemic crinkling symptoms were identified in Hainan Province in China in 2016. To survey and control the disease, it is necessary to identify and characterize the pathogen causing the disease. Dot enzyme-linked immunosorbent assay showed that the crude saps of the infected tomato samples reacted positively with the monoclonal antibody against Tobacco mosaic virus which indicated that one or more tobamoviruses are likely associated with the disease. RT-PCR and DNA sequence analysis results further elucidated that Tomato mottle mosaic virus (ToMMV) in Tobamovirus was the pathogen causing the mottle disease in tomato. We amplified and sequenced the full-length sequence of the genome which showed the highest nucleotide identity with ToMMV YYMLJ and ToMMV TiLhaLJ isolates. The putative virus isolate was named ToMMV Hainan. Biological indexing studies showed that ToMMV Hainan can infect Nicotiana benthamiana, Capsicum annuum and Solanum lycopersicum showing serious symptoms. This was the first identification and characterization of ToMMV infecting tomato in Hainan of China.

Apple (Malus domestica) fruits exhibiting bright stripe symptoms were identified in Weihai City, Shandong Province, China. To investigate the virome in the apple samples, the method of high throughput sequencing (HTS) was used to identify the viruses. It was found that the sequence of citrus concave gum-associated virus (CCGaV) was involved in the apple transcriptome dataset. The full-length genome of the CCGaV-Weihai isolate contained two segments, the RNA1 was 6674 nt in size containing a conserved RNA-dependent RNA polymerase (RdRp), and the RNA2 was ambisense, 2706 nt in length, encoding a movement protein (MP) and a coat protein (CP). Sequence alignment and phylogenetic analyses indicated that CCGaV-Weihai was more closely related to CCGaV-H2799 isolated from the apple host in the United States and distantly related to CCGaV-CGW2 from Citrus sinensis in Italy, indicating a possibly geographical and host differentiation of CCGaV isolates. This was the first identification and characterization of CCGaV infecting apples in China. Additionally, a rapid and sensitive reverse transcription recombinase polymerase amplification (RT-RPA) assay technique was established for CCGaV detection in apple plants. The RT-RPA of CCGaV was not affected by other common viruses in apple plants and is about 10-fold more sensitive than the conventional reverse transcription polymerase chain reaction method, which can be used in large-scale testing.