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The jasmonic acid (JA) pathway plays crucial roles in plant defence against pathogens and herbivores. Rice stripe virus (RSV) is the type member of the genus Tenuivirus. It is transmitted by the small brown planthopper (SBPH) and causes damaging epidemics in East Asia. The role(s) that JA may play in the tripartite interaction against RSV, its host, and vector are poorly understood. Here, we found that the JA pathway was induced by RSV infection and played a defence role against RSV. The coat protein (CP) was the major viral component responsible for inducing the JA pathway. Methyl jasmonate treatment attracted SBPHs to feed on rice plants while a JA‐deficient mutant was less attractive than wild‐type rice. SBPHs showed an obvious preference for feeding on transgenic rice lines expressing RSV CP. Our results demonstrate that CP is an inducer of the JA pathway that activates plant defence against RSV while also attracting SBPHs to feed and benefitting viral transmission. This is the first report of the function of JA in the tripartite interaction between RSV, its host, and its vector., Rice stripe virus coat protein is an inducer of the jasmonic acid pathway that activates plant defence against the virus while also attracting its vector to feed and benefitting viral transmission.

Background Nicotiana benthamiana is widely used as a model plant to study plant-pathogen interactions. Fasciclin-like arabinogalactan proteins (FLAs), a subclass of arabinogalactan proteins (AGPs), participate in mediating plant growth, development and response to abiotic stress. However, the members of FLAs in N. benthamiana and their response to plant pathogens are unknown. Results 38 NbFLAs were identified from a genome-wide study. NbFLAs could be divided into four subclasses, and their gene structure and motif composition were conserved in each subclass. NbFLAs may be regulated by cis-acting elements such as STRE and MBS, and may be the targets of transcription factors like C2H2. Quantitative real time polymerase chain reaction (RT-qPCR) results showed that selected NbFLAs were differentially expressed in different tissues. All of the selected NbFLAs were significantly downregulated following infection by turnip mosaic virus (TuMV) and most of them also by Pseudomonas syringae pv tomato strain DC3000 (Pst DC3000), suggesting possible roles in response to pathogenic infection. Conclusions This study systematically identified FLAs in N. benthamiana, and indicates their potential roles in response to biotic stress. The identification of NbFLAs will facilitate further studies of their role in plant immunity in N. benthamiana.

Yam bean (Pachyrhizus erosus), a high-yielding leguminous root crop with good nutritional value, is widely cultivated in southern China. In 2020, P. erosus (cv. Mumashan) plants exhibiting irregular yellow leaves and malformed seed pods (Supplementary Fig S1) were observed at Ningbo city, Zhejiang Province, China. To determine the causal agent(s) of the disease, symptomatic leaves (n=4) were collected for electron microscopy negative staining. Virus particles with a length of about 700nm, similar to viruses in the genus Potyvirus, were observed via transmission electron microscope (TEM), suggesting the presence a potyvirus(es). To further confirm which potyvirus(es) infected yam bean, total RNA was extracted from leaf samples of a total of six plants, including four symptomatic plants and two asymptomatic plants using TRIzol reagent (Invitrogen Carlsbad, CA, USA) according to the manufacturer's instructions. RNA was reverse-transcribed into cDNA with M4-T as the 3'-anchoring primer by ReverTra Ace® kit (Toyobo, Japan). Sprimer/M4 Potyviridae specific primers (Chen et al., 2001) were used for PCR analysis. A ~1,700-bp-long product was amplified from four symptomatic plants using KOD FX enzyme (Toyobo, Japan). No such band was amplified from the two asymptomatic plants. The PCR product (~1.7kb) amplified from a single symptomatic plant was ligated into the pEASY®-Blunt Zero vector (TransGen Bio, Beijing, China) and sequenced (Sangon Bio, Shanghai, China). The amplicon showed 99% nucleotide sequence identities with bean common mosaic virus (BCMV) isolate NKY021 (KJ807819). Subsequently, the complete nucleotide sequences of this BCMV isolate (referred as BCMV-NB) was amplified by overlapping RT-PCR and rapid amplification of cDNA ends with primers (Supplementary Table S1) designed from the sequence of BCMV isolate NKY021. The BCMV-NB full genome (Accession No. OL871237) consists of 10,053 nucleotides excluding the poly(A) tail and contains a large open reading frame encoding a polyprotein of 3222 amino acids. BLASTn analysis showed that BCMV-NB shared a sequence identity of 96.4% with BCMV isolate HZZB011 (KJ807815). Phylogenetic tree generated by Neighbour-Joining method revealing the BCMV-NB isolate was grouped together with Chinese isolates from Glycine max (Supplementary Fig S1). To test the infectivity of BCMV-NB, virus-free yam bean (cv. Mumashan) and Nicotiana benthamiana seedlings were mechanically inoculated with sap extracted from the symptomatic leaves of a BCMV-NB-infected yam bean plant. The inoculated yam bean plants developed typical BCMV mosaic and chlorotic symptoms at 16 days post inoculation (dpi), while Nicotiana benthamiana had no obvious symptoms at 10 or 20 dpi (Supplementary Fig S1). BCMV infections were confirmed in yam bean plants (infection rate 6/6) and N. benthamiana plants (infection rate 8/8) by RT-PCR at 16 dpi and 10 dpi, respectively. Twelve further P. erosus plants (cv. Mumashan) were collected from a field in Ningbo city and tested by RT-PCR with BCMV-specific primer pair BCMV CP (+)/(-) (Supplementary Table 1). Eight out of the 12 samples tested positive for BCMV by PCR-gel electrophoresis (Supplementary Fig S1) and Sanger sequencing, suggesting a high incidence of BCMV infection in this field. BCMV infection in yam bean has been reported from Indonesia (Damayanti et al., 2008) and Peru (Fuentes et al., 2012). To the best of our knowledge, this is the first report of BCMV naturally infecting yam bean in China. Thus, special attention and appropriate management strategies are needed to minimize the damage caused by BCMV to yam bean crops in China.

Ferredoxin 1 (FD1) accepts and distributes electrons in the electron transfer chain of plants. Its expression is universally downregulated by viruses and its roles in plant immunity have been brought into focus over the past decade. However, the mechanism by which viruses regulate FD1 remains to be defined. In a previous report, we found that the expression of Nicotiana benthamiana FD1 (NbFD1) was downregulated following infection with potato virus X (PVX) and that NbFD1 regulates callose deposition at plasmodesmata to play a role in defense against PVX infection. We now report that NbFD1 is downregulated by rice stripe virus (RSV) infection and that silencing of NbFD1 also facilitates RSV infection, while viral infection was inhibited in a transgenic line overexpressing NbFD1, indicating that NbFD1 also functions in defense against RSV infection. Next, a RSV-derived small interfering RNA was identified that contributes to the downregulation of FD1 transcripts. Further analysis showed that the abscisic acid (ABA) which accumulates in RSV-infected plants also represses NbFD1 transcription. It does this by stimulating expression of ABA insensitive 5 (ABI5), which binds the ABA response element motifs in the NbFD1 promoter, resulting in negative regulation. Regulation of FD1 by ABA was also confirmed in RSV-infected plants of the natural host rice. The results therefore suggest a mechanism by which virus regulates chloroplast-related genes to suppress their defense roles.

Jasmonic acid (JA) is a crucial hormone in plant antiviral immunity. Increasing evidence shows that viruses counter this host immune response by interfering with JA biosynthesis and signaling. However, the mechanism by which viruses affect JA biosynthesis is still largely unexplored. Here, we show that a highly conserved chloroplast protein cpSRP54 was downregulated in Nicotiana benthamiana infected by turnip mosaic virus (TuMV). Its silencing facilitated TuMV infection. Furthermore, cpSRP54 interacted with allene oxide cyclases (AOCs), key JA biosynthesis enzymes, and was responsible for delivering AOCs onto the thylakoid membrane (TM). Interestingly, TuMV P1 protein interacted with cpSRP54 and mediated its degradation via the 26S proteosome and autophagy pathways. The results suggest that TuMV has evolved a strategy, through the inhibition of cpSRP54 and its delivery of AOCs to the TM, to suppress JA biosynthesis and enhance viral infection. Interaction between cpSRP54 and AOCs was shown to be conserved in Arabidopsis and rice, while cpSRP54 also interacted with, and was degraded by, pepper mild mottle virus (PMMoV) 126 kDa protein and potato virus X (PVX) p25 protein, indicating that suppression of cpSRP54 may be a common mechanism used by viruses to counter the antiviral JA pathway., Author summary Jasmonic acid pathway has emerged as one of the predominant battlefields between plants and viruses. Several studies have indicated that, in addition to interfering with JA signaling, plant viruses can also affect JA biosynthesis, but the direct molecular links between them remain elusive. Here, we identify a highly conserved chloroplast protein cpSRP54 as a key positive regulator in JA biosynthesis and a common target for viruses belong to different genera. Through associating with cpSRP54 and inducing its degradation using the protein they encoded, the viruses can inhibit the cpSRP54-facilitated delivery of AOCs to the thylakoid membrane and manipulation of JA-mediated defense. This capability of viruses might define a novel and effective strategy against the antiviral JA pathway.

Pepper mild mottle virus (PMMoV) causes serious economic losses in pepper production in China. In a survey for viral diseases on pepper, two PMMoV isolates (named PMMoV-ZJ1 and PMMoV-ZJ2) were identified with different symptoms in Zhejiang province. Sequence alignment analysis suggested there were only four amino acid differences between the isolates: Val262Gly, Ile629Met and Ala1164Thr in the replicase, and Asp20Asn in the coat protein. Infectious cDNA clones of both isolates were constructed and shown to cause distinctive symptoms. Chlorosis symptoms appeared only on PMMoV-ZJ2-infected plants and the Asp20Asn substitution in the CP was shown to be responsible. Confocal assays revealed that the subcellular localization pattern of the two CPs was different, CP20Asp was mainly located at the cell periphery, whereas most CP20Asn located in the chloroplast. Thus, a single amino acid in the CP determined the chlorosis symptom, accompanied by an altered subcellular localization.

Gynura japonica (Thunb.) Juel [Asteraceae; syn: G. segetum (Lour.) Merr] is an important perennial medicinal herb used in China for topical treatment of trauma injuries (Lin et al. 2003). It grows naturally in the southern provinces of China and is also sometimes cultivated. During 2018-2020, wild G. japonica plants exhibiting chlorotic spots and mosaic symptoms were observed in Zhejiang province, China. To identify the possible causal agents of the disease, a single symptomatic leaf sample was collected in August 2019 and sent to Zhejiang Academy of Agricultural Sciences (Hangzhou, China) for next generation sequencing (NGS). Total RNAs extracted with TRIzol (Invitrogen, Carlsbad, USA) were subjected to high throughput sequencing on the Illumina NovaSeq 6000 platform with PE150bp and data analysis was performed by CLC Genomic Workbench 11 with default parameters (QIAGEN, Hilden, Germany). A total of 37,314,080 paired-end reads were obtained, and 11,785 contigs (961 to 10,964 bp) were generated and compared with sequences in GenBank using BLASTn or BLASTx. Of the total of 12 viral-related contigs obtained, one with a length of 6,442 nt mapped to the genomic RNA of ASGV (MN495979), seven contigs with lengths ranging from 1,034 to 2,901 nt mapped to Chrysanthemum virus B (CVB), and four mapped to broad bean wilt virus 2 (BBWV2), a virus which is known to infect G. procumbens (Kwak et al. 2017). To further confirm the presence of ASGV and CVB, primers were designed and the complete nucleotide sequences of both viruses were amplified from the original NGS sample using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) according to the manufacturer's instructions (Tiosbio, Beijing, China). BLASTn analysis revealed that the complete 6,451 nt sequence of ASGV (GenBank accession No. MW259059) shared the highest identity (81.2%) with a Chinese isolate of ASGV from citrus (MN495979). The two isolates grouped with another Chinese isolate (from pear) in phylogenetic analysis. The predicted coat protein of the virus had the highest nt identity of 93.7% (96.2% amino acid sequence identity) with that of the Chinese ASGV isolate XY from apple (KX686100). The complete genomes of two distinct molecular variants of CVB (both 8,987 nt in length) were also obtained from this sample (GenBank accession Nos. MW269552, MW269553). They shared 86.8% nt identity with each other and had 81.1% and 82.1% identity to the only known complete sequence of CVB from chrysanthemum (AB245142). Ten additional wild G. japonica plants with mosaic symptoms were collected randomly during 2019-2020 from Hangzhou (n=6) and Ningbo (n=4) in Zhejiang province and tested by RT-PCR with specific primer pairs to detect BBWV2, ASGV and CVB. RT-PCR and subsequent sequencing revealed that these three viruses were present in all the samples tested, indicating that co-infection of G. japonica by ASGV, CVB and BBWV2 is common. CVB mainly infects chrysanthemum (Singh et al. 2012), while ASGV is known as a pathogen of various fruit trees especially in the family Rosaceae, although there are recent reports that it can also infect some plants in Gramineae, Asparagaceae and Nelumbonaceae (Bhardwaj et al. 2017; Chen et al. 2019; He et al. 2019). Our results provide the first report that Gynura is a natural host of CVB and ASGV. Further surveys and biological studies are underway to evaluate the importance of Gynura as a virus reservoir for epidemics among the various hosts.

Lagenaria siceraria is an economically important cucurbitaceous crop, but suitable reference genes (RGs) to use when the plants are infected by cucumber green mottle mosaic virus (CGMMV) have not been determined. Sixteen candidate RGs of both leaf and fruit and 18 candidate RGs mostly from separate RNA-Seq datasets of bottle gourd leaf or fruit were screened and assessed by RT-qPCR. The expression stability of these genes was determined and ranked using geNorm, NormFinder, BestKeeper and RefFinder. Comprehensive analysis resulted in the selection of LsCYP, LsH3, and LsTBP as the optimal RGs for bottle gourd leaves, and LsP4H, LsADP, and LsTBP for fruits. LsWD, LsGAPDH, and LsH3 were optimal for use in both leaves and fruits under the infection of CGMMV. Isopentenyl transferase (IPT) and DNA-directed RNA polymerase (DdRP) were used to validate the applicability of the most stable identified RGs from bottle gourd in response to CGMMV. All the candidate RGs performed in RT-qPCR consistently with the data from the transcriptome database. The results demonstrated that LsWD, LsGAPDH and LsH3 were the most suitable internal RGs for the leaf, and LsH3, LsGAPDH, LsP4H and LsCYP for the fruit.

RNA silencing is an evolutionarily conserved antiviral mechanism, through which virus-derived small interfering RNAs (vsiRNAs) playing roles in host antiviral defence are produced in virus-infected plant. Deep sequencing technology has revolutionized the study on the interaction between virus and plant host through the analysis of vsiRNAs profile. However, comparison of vsiRNA profiles in different tissues from a same host plant has been rarely reported. In this study, the profiles of virus-derived small interfering RNAs (vsiRNAs) from leaves and fruits of Lagenaria siceraria plants infected with Cucumber green mottle mosaic virus (CGMMV) were comprehensively characterized and compared. Many more vsiRNAs were present in infected leaves than in fruits. vsiRNAs from both leaves and fruits were mostly 21- and 22-nt in size as previously described in other virus-infected plants. Interestingly, vsiRNAs were predominantly produced from the viral positive strand RNAs in infected leaves, whereas in infected fruits they were derived equally from the positive and negative strands. Many leaf-specific positive vsiRNAs with lengths of 21-nt (2,058) or 22-nt (3,996) were identified but only six (21-nt) and one (22-nt) positive vsiRNAs were found to be specific to fruits. vsiRNAs hotspots were only present in the 5’-terminal and 3’-terminal of viral positive strand in fruits, while multiple hotspots were identified in leaves. Differences in GC content and 5'-terminal nucleotide of vsiRNAs were also observed in the two organs. To our knowledge, this provides the first high-resolution comparison of vsiRNA profiles between different tissues of the same host plant.