Background N6-methyladenosine (m6A) is the most common RNA modification in eukaryotes and has been implicated as a novel epigenetic marker that is involved in various biological processes. The pattern and functional dissection of m6A in the regulation of several major human viral diseases have already been reported. However, the patterns and functions of m6A distribution in plant disease bursting remain largely unknown. Results We analyse the high-quality m6A methylomes in rice plants infected with two devastating viruses. We find that the m6A methylation is mainly associated with genes that are not actively expressed in virus-infected rice plants. We also detect different m6A peak distributions on the same gene, which may contribute to different antiviral modes between rice stripe virus or rice black-stripe dwarf virus infection. Interestingly, we observe increased levels of m6A methylation in rice plant response to virus infection. Several antiviral pathway-related genes, such as RNA silencing-, resistance-, and fundamental antiviral phytohormone metabolic-related genes, are also m6A methylated. The level of m6A methylation is tightly associated with its relative expression levels. Conclusions We revealed the dynamics of m6A modification during the interaction between rice and viruses, which may act as a main regulatory strategy in gene expression. Our investigations highlight the significance of m6A modifications in interactions between plant and viruses, especially in regulating the expression of genes involved in key pathways.

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Phosphorylation is one of the most extensively investigated post-translational modifications that orchestrate a variety of cellular signal transduction processes. The phosphorylation of virus-encoded proteins plays an important regulatory role in the infection cycle of such viruses in plants. In recent years, molecular mechanisms underlying the phosphorylation of plant viral proteins have been widely studied. Based on recent publications, our study summarizes the phosphorylation analyses of plant viral proteins and categorizes their effects on biological functions according to the viral life cycle. This review provides a theoretical basis for elucidating the molecular mechanisms of viral infection. Furthermore, it deepens our understanding of the biological functions of phosphorylation in the interactions between plants and viruses.

Rehmannia glutinosa is a top-grade traditional Chinese medicine, and also is an important planting medicinal material for Chinese poor farmers shaking off poverty. Rehmannia mosaic virus (ReMV) causes big economic loss of R. glutinosa in planting area. However, there is no effective methods for quick, accurate, and high-throughput detection for ReMV in Chinese production area. The preserved R. glutinosa samples carrying ReMV was taken for research material. The coat protein coding sequences (CPReMV) was cloned and sequenced. The target sequence was further placed into a prokaryotic expression vector to express the N-terminal-tagged recombinant CPReMV protein (His-CPReMV). Purified His-CPReMV was used as an antigen to immunize New Zealand white rabbits, and antiserum was obtained. The titers and sensitivities of the antisera were analyzed and evaluated. Polyclonal antibodies were purified from the antiserum, and the titers and sensitivity to the target His-CPReMV protein were evaluated. The results demonstrate that the obtained polyclonal antibodies against His-CPReMV could be successfully used for rapid, accurate, and high-throughput detection of ReMV from R. glutinosa planted in the wild. Our investigation established serological-based detection methods for ReMV for the first time, and provides a foundation for future exploration of the pathogenic mechanisms of ReMV in R. glutinosa.

Infectious cloning of plant viruses is a powerful tool for studying the reverse genetic manipulation of viral genes in virus–host plant interactions, contributing to a deeper understanding of the life history and pathogenesis of viruses. Yet, most of the infectious clones of RNA virus constructed in E. coli are unstable and toxic. Therefore, we modified the binary vector pCass4-Rz and constructed the ternary shuttle vector pCA4Y. The pCA4Y vector has a higher copy number in the E. coli than the conventional pCB301 vector, can obtain a high concentration of plasmid, and is economical and practical, so it is suitable for the construction of plant virus infectious clones in basic laboratories. The constructed vector can be directly extracted from yeast and transformed into Agrobacterium tumefaciens to avoid toxicity in E. coli. Taking advantage of the pCA4Y vector, we established a detailed large and multiple DNA HR-based cloning method in yeast using endogenous recombinase. We successfully constructed the Agrobacterium-based infectious cDNA clone of ReMV. This study provides a new choice for the construction of infectious viral clones.

Rehmannia glutinosa Libosch. is a perennial herbaceous plant of the family Scrophulariaceae. Its roots can be used as traditional Chinese medicine. The asexual reproduction by vegetative organ of R. glutinosa lead to an increased viral disease that seriously affects its yield and quality (Kwak et al. 2020; Kwak et al. 2018; Ling and Liu 2009). Leaves of R. glutinosa in Wenxian County, Henan Province, China showed symptoms of chlorosis, mosaic and irregular yellow in August 2019. In general, the older leaves at the base or middle of the plant (sample 2# and 5#) first became irregular yellowing, followed by a gradual extend to the leaves at the top (Supplementary Fig. S1A). Six plants (2#, 3#, 5#, 7#, 8#, and 9#) with these symptoms were collected. The total RNA was extracted and its siRNAs were obtained. High-throughput siRNA sequencing (Sangon, Shanghai, China) was performed on Illumina Hiseq 2000 platform with paired-end method after siRNA library construction (NEBNext Ultra II RNA Library Prep Kit, NEB, UK). Sequencing files were treated with Illumina’s CASAVA pipeline (version 1.8). The length of the resulting reads with adaptor removed were mostly distributed ranging from 21-24 nt (Supplementary Fig. S1B). The Velvet Software 0.7.31 (k=17) was taken to do de novo assembling, and the contigs (∼13,000, Contigs > 300 bp) were used to perform BLASTN against GenBank database. Two viruses, Rehmannia mosaic virus (ReMV) and cucurbit chlorotic yellows virus (CCYV), were frequently appeared in analyzed six symptomatic samples. To further identify the infection of CCYV to R. glutinosa, ten samples with virus-infected symptoms were randomly collected. Total protein and RNAs were extracted for RT-PCR and ELISA (HALING. Shanghai, China). A specific pair of primers (Supplementary Table S1) were designed to amplify the 753-bp length coat protein (CP) gene of CCYV. The result showed that two samples appeared a specific band of expected size on the agarose gel, which indicated that they were infected by CCYV (Supplementary Fig. S1C, Upper panel). The same result was obtained by ELISA assay (Supplementary Fig. S1D). The amplified CP fragment of CCYV was recycled and purified by TIANgel Midi Purification Kit (Tiangen, Beijing, China), followed by cloned into pMD19-T (TaKaRa, Dalian, China) and transformed into E. coli DH5a.Ten separate clones were selected and sequenced (Sangon, Shanghai, China) after PCR verification. The obtained sequences (GenBank accession No. MW521380 & MW521381) were analyzed by BLASTN and bioEdit software (version 7.2.3). The results showed 100% identity with the CCYV CP sequences that mainly derived from infected cucurbit. To confirm the occurrence and distribution of CCYV and ReMV in planting area, the other twenty-four samples (20 with chlorosis and stunt symptoms and 4 with invisible symptoms) were randomly collected for RT-PCR in different regions of Henan Province (Supplementary Table S1). The results showed that the CCYV and ReMV infection rate were 20.5% and 61.7%, respectively. Co-infection of the CCYV and ReMV was 5.8% in fields (Supplementary Table S2). In sum, these results indicated the CCYV can naturally infect R. glutinosa in China. CCYV is transmitted by white-fly in a semi-persistent manner and mainly damages cucurbits (Orfanidou et al. 2017). CCYV has been discovered in many places (Huang et al. 2010). To date, there is no report about CCYV infecting R. glutinosa in nature. This is the first report of CCYV naturally infect R. glutinosa in China.

Cowpea (Vigna unguiculata) is a crop grown worldwide as a protein source for food and feed (Lonardi et al. 2019). During the summer of 2019, noticeable virus-like symptoms such as mosaic, leaf narrowing, stunt and chlorosis were observed on cowpeas ‘Xianfeng’ planted in Yangzhou city and its suburbs, Jiangsu Province, East China (Supplementary Fig. S1A). The total RNA was extracted from both symptomatic and asymptomatic plants by RNAiso Plus (TaKaRa, Dalian, China) and sRNAs were separated and recovered by gel purification. The NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, UK) was used for sRNA library construction. The library was sequenced with the paired-end method on the Illumina Hiseq 2000 platform (Sangon, Shanghai, China). The obtained sequencing files were treated with Illumina’s CASAVA pipeline (version 1.8). The reads resulting from sequencing were further processed with adaptor removing, and the most abundant sRNAs were distributed from 21-24 nt (Supplementary Fig. S1B). The de novo assembly was performed with the Velvet Software 0.7.31 (k=17), and the obtained contigs (∼12,000, Contigs > 500 bp) were used perform a BLAST search against the GenBank viral reference database. Fifteen contigs with high similarities of 98.61% to 99.64% and coverage of 94% to the reported vicia cryptic virus M (VCV-M) genomic sequence (GenBank accession No. EU371896) were identified. Other common viruses, such as cowpea mosaic virus (CPMV), cowpea aphid-borne mosaic virus (CABMV), and cucumber mosaic virus (CMV), were also included (Unpublished).VCV-M belongs to the genus Amalgavirus, family Amalgaviridae (Nibert et al. 2016). Amalgaviruses are efficiently transmitted through seeds but not mechanically or by grafting (Sabanadzovic et al. 2009). To confirm the presence of VCV-M in the collected plants, total RNA was isolated and the first-strand cDNA was prepared by M-MLV reverse transcriptase (TaKaRa, Dalian, China) using specific primers. Primers (Supplementary Table SI) were designed according to the assembled contigs. Polymerase chain reaction (PCR) was performed to amplify the targeted genomic fragment of VCV-M, and the predicted 3,434 bp amplicon was obtained from five cowpea plants (Supplementary Fig. S1C). A randomly selected amplicon was purified with the TIANgel Midi Purification Kit (Tiangen, Beijing, China) and cloned to pMD19-T (TaKaRa, Dalian, China) for sequencing (Sangon, Shanghai, China). The obtained consensus sequence (GenBank accession No. MN015673) was analyzed and showed 99.39% similarity with the reported VCV-M genome (GenBank accession No. EU371896). To confirm the occurrence and distribution of VCV-M infection, 17 cowpea samples of different cultivars (4 with yellowing and stunt symptoms and 13 without visible symptoms) were collected from different regions of Jiangsu Province and tested using RT-PCR with specific primers (Supplementary Fig. S1C). They were further tested by western blot (WB) detection as described previously (Zhang et al. 2017). Specific CPVCV-M antiserum was obtained by immunizing the New Zealand white rabbits with the prokaryotic expressed recombinant His-CPVCV-M protein (HuaBio, Hangzhou, China). WB results (Supplementary Fig. S1D) and RT-PCR resulted in five samples that were positive out of a total of 17 samples, suggesting the VCV-M infection is common in cowpea plants. To determine whether the VCV-M was the causal agent or contributor to the observed symptoms, we investigated the presence of other cowpea-infecting viruses (CPMV, CABMV, and CMV) in these samples through RT-PCR with specific primers for each virus (Supplementary Table SI) and ELISA with commercial kits. RT-PCR and ELISA detection results showed mixed infection by VCV-M/CPMV (n = 1), VCV-M/CABMV (n = 1), VCV-M/CMV (n = 1), or VCV-M/CPMV/CABMV/CMV (n = 2). The VCV-M/CABMV co-infected sample was asymptomatic. Taken together, the symptoms on cowpea could not be attributed to one particular viral infection. To further confirm VCV-M infection, we selected four samples (two positive and two negative, as determined by RT-PCR and WB) for northern blot assay. The probe was prepared with the DIG Random Labeling and Detection Kit I (POD) for color detection with DAB (BOSTER, Wuhan, China). The Northern blot assay was performed as previously described with minor modifications (Prosniak et al. 2001). The results (Supplementary Fig. S1E) confirmed the accuracy of previous RT-PCR and WB analyses. To our knowledge, this is the first report of VCV-M infection of cowpea plants in China. Although it is commonly accepted that VCV-M causes no symptoms, the roles of such viruses in affecting their hosts’ biological characteristics, which are often influenced by co-infection conditions, remains unclear.