The sugarcane streak mosaic virus (SCSMV) is the most important disease in sugarcane produced in southern China. The SCSMV encoded protein 1 (P1SCSMV) is important in disease development, but little is known about its detailed functions in plant–virus interactions. Here, the differential accumulated proteins (DAPs) were identified in the heterologous expression of P1SCSMV via a potato virus X (PVX)-based expression system, using a newly developed four-dimensional proteomics approach. The data were evaluated for credibility and reliability using qRT-RCR and Western blot analyses. The physiological response caused by host factors that directly interacted with the PVX-encoded proteins was more pronounced for enhancing the PVX accumulation and pathogenesis in Nicotiana benthamiana. P1SCSMV reduced photosynthesis by damaging the photosystem II (PSII). Overall, P1SCSMV promotes changes in the physiological status of its host by up- or downregulating the expression of host factors that directly interact with the viral proteins. This creates optimal conditions for PVX replication and movement, thereby enhancing its accumulation levels and pathogenesis. Our investigation is the first to supply detailed evidence of the pathogenesis-enhancing role of P1SCSMV, which provides a deeper understanding of the mechanisms behind virus–host interactions.

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.

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.

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.

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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.

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