Transgenic expression of artificial microRNA targeting soybean mosaic virus P1 gene confers virus resistance in plant.

RNA silencing is an innate immune mechanism of plants against invasion by viral pathogens. Artificial microRNA (amiRNA) can be engineered to specifically induce RNA silencing against viruses in transgenic plants and has great potential for disease control. Here, we describe the development and application of amiRNA-based technology to induce resistance to soybean mosaic virus (SMV), a plant virus with a positive-sense single-stranded RNA genome. We have shown that the amiRNA targeting the SMV P1 coding region has the highest antiviral activity than those targeting other SMV genes in a transient amiRNA expression assay. We transformed the gene encoding the P1-targeting amiRNA and obtained stable transgenic Nicotiana benthamiana lines (amiR-P1-3-1-2-1 and amiR-P1-4-1-2-1). Our results have demonstrated the efficient suppression of SMV infection in the P1-targeting amiRNA transgenic plants in an expression level-dependent manner. In particular, the amiR-P1-3-1-2-1 transgenic plant showed high expression of amiR-P1 and low SMV accumulation after being challenged with SMV. Thus, a transgenic approach utilizing the amiRNA technology appears to be effective in generating resistance to SMV.

Generation of a Triple-Shuttling Vector and the Application in Plant Plus-Strand RNA Virus Infectious cDNA Clone Construction.

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.

Natural occurrence of broad bean wilt virus 2 on Mirabilis jalapa in China.

Mirabilis jalapa Libosch. is an annual ornamental herbaceous plant. Its leaves and roots are used as a traditional folk medicine that function in clearing heat and detoxifying, promoting blood circulation, regulating menstruation, and nourishing kidney (Annapoorani et al. 2014; Liu et al. 2020; Wang et al. 2018). Broad bean wilt virus 2 (BBWV-2), which belongs to the family Secoviridae, is transmitted by aphid in a non-persistent manner in the nature (Kondo et al. 2005) and mainly damages Vicia faba, pepper, yam and spinach (He et al. 2021). The leaves of M. jalapa on the campus showed shrinking (Supplementary Fig. 1A), yellowing (Supplementary Fig. 1B), mosaic (Supplementary Fig. 1D & 1E), and the whole plant had stunted and rough (Supplementary Fig. 1A & 1C) symptoms in the autumn of 2021. Eight plants (S21-S28) with these symptoms were harvested for total RNA extraction, siRNA mixture purification, and siRNA library made (NEBNext® Ultra™ II RNA Library Prep Kit for Illumina®, NEB, UK). The high-throughput siRNA sequencing with pair-end method was performed on Illumina Hiseq 2000 platform (Sangon, Shanghai, China). The raw sequencing data was treated with the Illumina's CASAVA pipeline (version 1.8). The adaptor was removed and the reads were mostly distributed in 21-24 nt length area (Supplementary Fig. 2A). The contigs (∼12,500, Length > 350 bp) were obtained by de novo assembling with the Velvet Software 0.7.31 (k = 17), then the BLASTN was preformed against GenBank database. Surprisingly, 237 contigs showed significant nucleotide sequence similarities to the genome of BBWV-2. To determine the incidence of BBWV-2 to M. jalapa in campus garden, twenty-eight leaf samples were randomly collected from the garden. Leave extract and total RNA of the sample were tested for BBWV-2 by ELISA (Agdia, USA, SRA46202/0096) and RT-PCR assay, respectively. Twenty-two samples were infected compared with the positive control, and their readings of ELISA were above or parallel to the positive control (Supplementary Fig. 2B∼2D). The coding sequence (1,395 bp) of BBWV-2 movement protein (MP) was amplified by a specific pair of primers (Supplementary Table S1) according to the contigs, the results indicated that the 22 out of 28 samples (78.6%) tested positive for BBWV-2 by both ELISA and RT-PCR (Supplementary Fig. 2E). The MP fragment of BBWV-2 obtained from one of the sample was purified by TIANgel Midi Purification Kit (Tiangen, Beijing, China) and then cloned into pMD19-T (TaKaRa, Dalian, China) vector. Ten separate clones were selected and sequenced (Sangon, Shanghai, China) after PCR verification. The obtained sequences (GenBank accession No. OM416039) were analyzed by BLASTN and bioEdit software (version 7.2.3). According to the phylogenetic tree constructed by BBWV-2 MP sequences (Supplementary Fig. 3), the obtained MP sequences (OM416039, ON677747, and ON677748) were most related to the BBWV-2 MP sequences that from pepper (GenBank accession No. JX183228.1), they share the nucleotide identity of 84.87%. To determine the occurrence and distribution of BBWV-2 in other areas, another twenty-two samples were randomly collected for RT-PCR in different regions of Jiangsu Province, China (Supplementary Table S2). The BBWV-2 infection rate was 76.0% in the M. jalapa. In sum, this is the first report of BBWV-2 naturally infecting M. Jalapa in China.

Phosphorylation of plant virus proteins: Analysis methods and biological functions.

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.

Development of polyclonal antibodies-based serological methods and a DIG-labelled DNA probe-based molecular method for detection of the Vicia cryptic virus-M in field plants.

Vicia cryptic virus M (VCV-M), a member of the genus Amalgavirus of the family Amalgaviridae, was first identified in 2009 in a Vicia faba Linn. planting in Hangzhou, Zhejiang Province, China. However, there has been no further research on the biological features of VCV-M to date and the viral particles and coat protein (CP) have not been identified. The putative CP of VCV-M was predicted from the viral genomic RNA. In this study, a recombinant version of the putative CP of VCV-M (His-CPVCV-M) was produced and used to prepare a polyclonal antiserum against the His-CPVCV-M. Using this antiserum, a Western blot, an immuno-dot-blot and an enzyme-linked immunosorbent assay were developed for testing field samples of V. faba for the presence of VCV-M. Additionally, a digoxigenin (DIG)-labelled DNA probe-based Northern blot assay was established for VCV-M genome detection in field samples. The results showed that both the serological and nucleic acid assays could accurately and sensitively detect VCV-M in V. faba. This research represented the first confirmed expression of the putative CP of VCV-M in infected V. faba tissues. The serological and nucleic acid assays provided two complementary methods for VCV-M detection which could contribute to seed quality control and production increases of V. faba crops.

The dynamics of N6-methyladenine RNA modification in interactions between rice and plant viruses.

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.

Cucurbit chlorotic yellows virus infecting Rehmannia glutinosa was detected in China.

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.

First report of vicia cryptic virus M infecting cowpea (Vigna unguiculata) 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.