Investigating the Interactions of the Cucumber Mosaic Virus 2b Protein with the Viral 1a Replicase Component and the Cellular RNA Silencing Factor Argonaute 1.

The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b protein's host targets is the RNA silencing factor Argonaute 1 (AGO1), which it binds to and inhibits. In Arabidopsis thaliana, if 2b-induced inhibition of AGO1 is too efficient, it induces reinforcement of antiviral silencing by AGO2 and triggers increased resistance against aphids, CMV's insect vectors. These effects would be deleterious to CMV replication and transmission, respectively, but are moderated by the CMV 1a protein, which sequesters sufficient 2b protein molecules into P-bodies to prevent excessive inhibition of AGO1. Mutant 2b protein variants were generated, and red and green fluorescent protein fusions were used to investigate subcellular colocalization with AGO1 and the 1a protein. The effects of mutations on complex formation with the 1a protein and AGO1 were investigated using bimolecular fluorescence complementation and co-immunoprecipitation assays. Although we found that residues 56-60 influenced the 2b protein's interactions with the 1a protein and AGO1, it appears unlikely that any single residue or sequence domain is solely responsible. In silico predictions of intrinsic disorder within the 2b protein secondary structure were supported by circular dichroism (CD) but not by nuclear magnetic resonance (NMR) spectroscopy. Intrinsic disorder provides a plausible model to explain the 2b protein's ability to interact with AGO1, the 1a protein, and other factors. However, the reasons for the conflicting conclusions provided by CD and NMR must first be resolved.

The non-template functions of helper virus RNAs create optimal replication conditions to enhance the proliferation of satellite RNAs.

As a type of parasitic agent, satellite RNAs (satRNAs) rely on cognate helper viruses to achieve their replication and transmission. During the infection of satRNAs, helper virus RNAs serve as templates for synthesizing viral proteins, including the replication proteins essential for satRNA replication. However, the role of non-template functions of helper virus RNAs in satRNA replication remains unexploited. Here we employed the well-studied model that is composed of cucumber mosaic virus (CMV) and its associated satRNA. In the experiments employing the CMV trans-replication system, we observed an unexpected phenomenon the replication proteins of the mild strain LS-CMV exhibited defective in supporting satRNA replication, unlike those of the severe strain Fny-CMV. Independent of translation products, all CMV genomic RNAs could enhance satRNA replication, when combined with the replication proteins of CMV. This enhancement is contingent upon the recruitment and complete replication of helper virus RNAs. Using the method developed for analyzing the satRNA recruitment, we observed a markedly distinct ability of the replication proteins from both CMV strains to recruit the positive-sense satRNA-harboring RNA3 mutant for replication. This is in agreement with the differential ability of both 1a proteins in binding satRNAs in plants. The discrepancies provide a convincing explanation for the variation of the replication proteins of both CMV strains in replicating satRNAs. Taken together, our work provides compelling evidence that the non-template functions of helper virus RNAs create an optimal replication environment to enhance satRNA proliferation.

A single amino acid at position 31 in the N-terminus of the coat protein of cucumber mosaic virus determines its avirulence function for RCY1-conferred virus resistance.

The coat protein (CP) of the cucumber mosaic virus (CMV) yellow strain [CMV(Y)], but not the CMV B2 strain [CMV(B2)], serves as an avirulence determinant against the NB-LRR class RCY1 of Arabidopsis thaliana. To investigate the avirulence function, a series of binary vectors were constructed by partially exchanging the CP coding sequence between CMV(Y) and CMV(B2) or introducing nucleotide substitutions. These vectors were transiently expressed in Nicotiana benthamiana leaves transformed with modified RCY1 cDNA. Analysis of hypersensitive resistance-cell death (HCD), CP accumulation, and defense gene expression at leaf sites infiltrated with Agrobacterium indicated that a single amino acid at position 31 of the CP seems to determine the avirulence function.

Cucumber mosaic virus impairs the physiological homeostasis of Panax notoginseng and induces saponin-mediated resistance.

As an important medicinal plant, Panax notoginseng often suffers from various abiotic and biotic stresses during its growth, such as drought, heavy metals, fungi, bacteria and viruses. In this study, the symptom and physiological parameters of cucumber mosaic virus (CMV)-infected P. notoginseng were analyzed and the RNA-seq was performed. The results showed that CMV infection affected the photosynthesis of P. notoginseng, caused serious oxidative damage to P. notoginseng and increased the activity of several antioxidant enzymes. Results of transcriptome analysis and corresponding verification showed that CMV infection changed the expression of genes related to plant defense and promoted the synthesis of P. notoginseng saponins to a certain extent, which may be defensive ways of P. notoginseng against CMV infection. Furthermore, pretreatment plants with saponins reduced the accumulation of CMV. Thus, our results provide new insights into the role of saponins in P. notoginseng response to virus infection.

Absence of Seed-Mediated Transmission of Cucumber Mosaic Virus in Espelette Pepper Crops despite Widespread and Recurrent Epidemics.

In the past decade, severe epidemics of cucumber mosaic virus (CMV) have caused significant damage to Espelette pepper crops. This virus threatens the production of Espelette pepper, which plays a significant role in the local economy and touristic attractiveness of the French Basque Country, located in southwestern France. In 2021 and 2022, CMV was detected via double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) in Gorria pepper seed lots harvested from naturally infected fields scattered throughout the entire Espelette pepper production area. These seed lots were used in greenhouse grow-out tests to determine whether CMV could be transmitted to seedlings from contaminated seeds, using visual symptom assessment, DAS-ELISAs, and reverse transcription-polymerase chain reaction (RT-PCR). Despite the widespread occurrence of CMV in seeds of field samples, the grow-out experiments on a total of over 5000 seedlings yielded no evidence of seed transmission of local CMV isolates in Gorria pepper. Therefore, rather than seeds from infected pepper plants, sources of CMV inoculum in Espelette are more likely to be alternative hosts present in and around pepper fields that can allow for the survival of CMV during the off-season. These results have important epidemiological implications and will guide the choice of effective measures to control current epidemics.

Identification of Host Factors Interacting with a γ-Shaped RNA Element from a Plant Virus-Associated Satellite RNA.

Previously, we identified a highly conserved, γ-shaped RNA element (γRE) from satellite RNAs of cucumber mosaic virus (CMV), and we determined γRE to be structurally required for satRNA survival and the inhibition of CMV replication. It remains unknown how γRE biologically functions. In this work, pull-down assays were used to screen candidates of host factors from Nicotiana benthamiana plants using biotin-labeled γRE as bait. Nine host factors were found to interact specifically with γRE. Then, all of these host factors were down-regulated individually in N. benthamiana plants via tobacco rattle virus-induced gene silencing and tested with infection by GFP-expressing CMV (CMV-gfp) and the isolate T1 of satRNA (sat-T1). Out of nine candidates, three host factors, namely histone H3, GTPase Ran3, and eukaryotic translation initiation factor 4A, were extremely important for infection by CMV-gfp and sat-T1. Moreover, we found that cytosolic glyceraldehyde-3-phosphate dehydrogenase 2 contributed to the replication of CMV and sat-T1, but also negatively regulated CMV 2b activity. Collectively, our work provides essential clues for uncovering the mechanism by which satRNAs inhibit CMV replication.

[Analysis of the Complete Tomato Aspermy Virus Genomes Suggests Reassortment in Russian Isolates from Chrysanthemum].

Tomato aspermy virus (TAV, genus Cucumovirus from the family Bromoviridae) is one of the most common and harmful chrysanthemum viruses, causing severe flower distortion, size reduction, and color breaking. Metatranscriptome sequencing of chrysanthemum plants of the Ribonette and Golden Standard cultivars from the collection of the Nikita Botanical Garden (Yalta, Republic of Crimea) generated TAV-related RNA reads. The complete genomes of two Russian isolates of the virus were assembled from the reads. This is the first report of full-length TAV genomes from Russia. Typically of cucumoviruses, the segmented TAV genome is represented by three single-stranded positive-sense linear RNA molecules of 3412 (RNA1), 3097 (RNA2) and 2219 (RNA3) nucleotides. Five open reading frames (ORF) have been identified that encode replicase (ORF1), RNA-dependent RNA polymerase (ORF2a), silencing suppressor protein (OFR2b), movement protein (OFR3a) and the coat protein (ORF3b). The identity of TAV genomes from the two chrysanthemum cultivars was 99.8% for all three viral RNAs; with other TAV isolates from GenBank it was 97.5-99.7% (RNA1), 93.8-99.8% (RNA2), and 89.3-99.3% (RNA3). Phylogenetic analysis showed that RNA1 and RNA3 of the Russian isolates were assigned to heterogeneous groups of TAV isolates found on various plant species in different regions of the world. At the same time, RNA2 clearly clustered with tomato isolates SKO20ST2 from Slovenia and PV-0220 from Bulgaria and, to a lesser extent, with the Iranian isolate Ker.Mah.P from petunia and the Chinese isolate Henan from chrysanthemum. The incongruence of phylogenetic trees reconstructed from different genome segments suggests pseudo-recombination (reassortment) in the Russian TAV isolates.

cmv1-Mediated Resistance to CMV in Melon Can Be Overcome by Mixed Infections with Potyviruses.

Resistance to cucumber mosaic virus (CMV) strain LS in melon is controlled by the gene cmv1, which restricts phloem entry. In nature, CMV is commonly found in mixed infections, particularly with potyviruses, where a synergistic effect is frequently produced. We have explored the possibility that this synergism could help CMV-LS to overcome cmv1-mediated resistance. We demonstrate that during mixed infection with a potyvirus, CMV-LS is able to overcome cmv1-controlled resistance and develop a systemic infection and that this ability does not depend on an increased accumulation of CMV-LS in mechanically inoculated cotyledons. Likewise, during a mixed infection initiated by aphids, the natural vector of both cucumoviruses and potyviruses that can very efficiently inoculate plants with a low number of virions, CMV-LS also overcomes cmv1-controlled resistance. This indicates that in the presence of a potyvirus, even a very low amount of inoculum, can be sufficient to surpass the resistance and initiate the infection. These results indicate that there is an important risk for this resistance to be broken in nature as a consequence of mixed infections, and therefore, its deployment in elite cultivars would not be enough to ensure a long-lasting resistance.

VIGS as a strategy to reverse aphid wing induction by Y-satellite RNA of cucumber mosaic virus.

Y-satellite RNA (Y-sat) of cucumber mosaic virus upregulates the expression of the aphid ABCG4 gene, which promotes aphid wing formation. We used ABCG4 virus-induced gene silencing (VIGS) to prevent the wing-induction mechanism of Y-sat and thus inhibited aphid wing formation. Of the aphids on plants with VIGS of ABCG4, only about 30% had wings, and 60-70% of the winged aphids were small and likely impaired in flying ability. In addition, we showed that double-stranded RNAs (dsRNAs) and small RNAs were transferred from the plant to the aphid to adequately silence aphid genes. Supplying ABCG4 dsRNA by VIGS to aphids is thus a potential strategy to inhibit aphid wing formation.

Argonaute 5-mediated antiviral defense and viral counter-defense in Nicotiana benthamiana.

The argonaute (AGO) family proteins play a crucial role in preventing viral invasions through the plant antiviral RNA silencing pathway, with distinct AGO proteins recruited for specific antiviral mechanisms. Our previous study revealed that Nicotiana benthamiana AGO5 (NbAGO5) expression was significantly upregulated in response to bamboo mosaic virus (BaMV) infection. However, the roles of NbAGO5 in antiviral mechanisms remained to be explored. In this research, we examined the antiviral functions of NbAGO5 in the infections of different viruses. It was found that the accumulation of NbAGO5 was induced not only at the RNA but also at the protein level following the infections of BaMV, potato virus X (PVX), tobacco mosaic virus (TMV), and cucumber mosaic virus (CMV) in N. benthamiana. To explore the antiviral mechanism and regulatory function of NbAGO5, we generated NbAGO5 overexpression (OE-NbAGO5) and knockout (nbago5) transgenic N. benthamiana lines. Our findings reveal that NbAGO5 provides defense against BaMV, PVX, TMV, and a mutant CMV deficient in 2b gene, but not against the wild-type CMV and turnip mosaic virus (TuMV). Through affinity purification and small RNA northern blotting, we demonstrated that NbAGO5 exerts its antiviral function by binding to viral small interfering RNAs (vsiRNAs). Moreover, we observed that CMV 2b and TuMV HC-Pro interact with NbAGO5, triggering its degradation via the 26S proteasome and autophagy pathways, thereby allowing these viruses to overcome NbAGO5-mediated defense. In addition, TuMV HC-Pro provides another line of counter-defense by interfering with vsiRNA binding by NbAGO5. Our study provides further insights into the antiviral RNA interference mechanism and the complex interplay between NbAGO5 and plant viruses.

A selective autophagy receptor VISP1 induces symptom recovery by targeting viral silencing suppressors.

Selective autophagy is a double-edged sword in antiviral immunity and regulated by various autophagy receptors. However, it remains unclear how to balance the opposite roles by one autophagy receptor. We previously identified a virus-induced small peptide called VISP1 as a selective autophagy receptor that facilitates virus infections by targeting components of antiviral RNA silencing. However, we show here that VISP1 can also inhibit virus infections by mediating autophagic degradation of viral suppressors of RNA silencing (VSRs). VISP1 targets the cucumber mosaic virus (CMV) 2b protein for degradation and attenuates its suppression activity on RNA silencing. Knockout and overexpression of VISP1 exhibit compromised and enhanced resistance against late infection of CMV, respectively. Consequently, VISP1 induces symptom recovery from CMV infection by triggering 2b turnover. VISP1 also targets the C2/AC2 VSRs of two geminiviruses and enhances antiviral immunity. Together, VISP1 induces symptom recovery from severe infections of plant viruses through controlling VSR accumulation.

Induction of aphid resistance in tobacco by the cucumber mosaic virus CMV∆2b mutant is jasmonate-dependent.

Cucumber mosaic virus (CMV) is vectored by aphids, including Myzus persicae. Tobacco (Nicotiana tabacum 'Xanthi') plants infected with a mutant of the Fny strain of CMV (Fny-CMVΔ2b, which cannot express the CMV 2b protein) exhibit strong resistance against M. persicae, which is manifested by decreased survival and reproduction of aphids confined on the plants. Previously, we found that the Fny-CMV 1a replication protein elicits aphid resistance in plants infected with Fny-CMVΔ2b, whereas in plants infected with wild-type Fny-CMV this is counteracted by the CMV 2b protein, a counterdefence protein that, among other things, inhibits jasmonic acid (JA)-dependent immune signalling. We noted that in nontransformed cv. Petit Havana SR1 tobacco plants aphid resistance was not induced by Fny-CMVΔ2b, suggesting that not all tobacco varieties possess the factor(s) with which the 1a protein interacts. To determine if 1a protein-induced aphid resistance is JA-dependent in Xanthi tobacco, transgenic plants were made that expressed an RNA silencing construct to diminish expression of the JA co-receptor CORONATINE-INSENSITIVE 1. Fny-CMVΔ2b did not induce resistance to M. persicae in these transgenic plants. Thus, aphid resistance induction by the 1a protein requires JA-dependent defensive signalling, which is countered by the CMV 2b protein.

Evaluation of sequencing and PCR-based methods for the quantification of the viral genome formula.

Viruses show great diversity in their genome organization. Multipartite viruses package their genome segments into separate particles, most or all of which are required to initiate infection in the host cell. The benefits of such seemingly inefficient genome organization are not well understood. One hypothesised benefit of multipartition is that it allows for flexible changes in gene expression by altering the frequency of each genome segment in different environments, such as encountering different host species. The ratio of the frequency of segments is termed the genome formula (GF). Thus far, formal studies quantifying the GF have been performed for well-characterised virus-host systems in experimental settings using RT-qPCR. However, to understand GF variation in natural populations or novel virus-host systems, a comparison of several methods for GF estimation including high-throughput sequencing (HTS) based methods is needed. Currently, it is unclear how HTS-methods compare a golden standard, such as RT-qPCR. Here we show a comparison of multiple GF quantification methods (RT-qPCR, RT-digital PCR, Illumina RNAseq and Nanopore direct RNA sequencing) using three host plants (Nicotiana tabacum, Nicotiana benthamiana, and Chenopodium quinoa) infected with cucumber mosaic virus (CMV), a tripartite RNA virus. Our results show that all methods give roughly similar results, though there is a significant method effect on genome formula estimates. While the RT-qPCR and RT-dPCR GF estimates are congruent, the GF estimates from HTS methods deviate from those found with PCR. Our findings emphasize the need to tailor the GF quantification method to the experimental aim, and highlight that it may not be possible to compare HTS and PCR-based methods directly. The difference in results between PCR-based methods and HTS highlights that the choice of quantification technique is not trivial.

Mutations in CmVPS41 controlling resistance to cucumber mosaic virus display specific subcellular localization.

Resistance to cucumber mosaic virus (CMV) in melon (Cucumis melo L.) has been described in several exotic accessions and is controlled by a recessive resistance gene, cmv1, that encodes a vacuolar protein sorting 41 (CmVPS41). cmv1 prevents systemic infection by restricting the virus to the bundle sheath cells, preventing viral phloem entry. CmVPS41 from different resistant accessions carries two causal mutations, either a G85E change, found in Pat-81 and Freeman's cucumber, or L348R, found in PI161375, cultivar Songwhan Charmi (SC). Here, we analyzed the subcellular localization of CmVPS41 in Nicotiana benthamiana and found differential structures in resistant and susceptible accessions. Susceptible accessions showed nuclear and membrane spots and many transvacuolar strands, whereas the resistant accessions showed many intravacuolar invaginations. These specific structures colocalized with late endosomes. Artificial CmVPS41 carrying individual mutations causing resistance in the genetic background of CmVPS41 from the susceptible variety Piel de Sapo (PS) revealed that the structure most correlated with resistance was the absence of transvacuolar strands. Coexpression of CmVPS41 with viral movement proteins, the determinant of virulence, did not change these localizations; however, infiltration of CmVPS41 from either SC or PS accessions in CMV-infected N. benthamiana leaves showed a localization pattern closer to each other, with up to 30% cells showing some membrane spots in the CmVPS41SC and fewer transvacuolar strands (reduced from a mean of 4 to 1-2) with CmVPS41PS. Our results suggest that the distribution of CmVPS41PS in late endosomes includes transvacuolar strands that facilitate CMV infection and that CmVPS41 re-localizes during viral infection.

Alpha-momorcharin preserves catalase activity to inhibit viral infection by disrupting the 2b-CAT interaction in Solanum lycopersicum.

Many host factors of plants are used by viruses to facilitate viral infection. However, little is known about how alpha-momorcharin (αMMC) counters virus-mediated attack strategies in tomato. Our present research revealed that the 2b protein of cucumber mosaic virus (CMV) directly interacted with catalases (CATs) and inhibited their activities. Further analysis revealed that transcription levels of catalase were induced by CMV infection and that virus accumulation increased in CAT-silenced or 2b-overexpressing tomato plants compared with that in control plants, suggesting that the interaction between 2b and catalase facilitated the accumulation of CMV in hosts. However, both CMV accumulation and viral symptoms were reduced in αMMC transgenic tomato plants, indicating that αMMC engaged in an antiviral role in the plant response to CMV infection. Molecular experimental analysis demonstrated that αMMC interfered with the interactions between catalases and 2b in a competitive manner, with the expression of αMMC inhibited by CMV infection. We further demonstrated that the inhibition of catalase activity by 2b was weakened by αMMC. Accordingly, αMMC transgenic plants exhibited a greater ability to maintain redox homeostasis than wild-type plants when infected with CMV. Altogether, these results reveal that αMMC retains catalase activity to inhibit CMV infection by subverting the interaction between 2b and catalase in tomato.

Genetic diversity and molecular characterization of Cucumber mosaic cucumovirus (CMV) subgroup II infecting Spinach (Spinacia oleracea) and Pea (Pisum sativum) in Pothwar region of Pakistan / Diversidade genética e caracterização molecular do Cucumber mosaic cucumovirus (CMV) subgrupo II infectando espinafre (Spinacia oleracea) e ervilha (Pisum sativum) na região de Pothwar, Paquistão

Abstract Cucumber mosaic virus (CMV) is a tremendous threat to vegetables across the globe, including in Pakistan. The present work was conducted to investigate the genetic variability of CMV isolates infecting pea and spinach vegetables in the Pothwar region of Pakistan. Serological-based surveys during 2016-2017 revealed 31.70% overall CMV disease incidence from pea and spinach crops. Triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) revealed that all the positive isolates belong to CMV subgroup II. Two selected cDNA from ELISA-positive samples representing each pea and spinach crops were PCR-amplified (ca.1100 bp) and sequenced corresponding to the CMV CP gene which shared 93.7% nucleotide identity with each other. Both the sequences of CMV pea (AAHAP) and spinach (AARS) isolates from Pakistan were submitted to GenBank as accession nos. MH119071 and MH119073, respectively. BLAST analysis revealed 93.4% sequence identity of AAHAP isolate with SpK (KC763473) from Iran while AARS isolate shared maximum identity (94.5%) with the strain 241 (AJ585519) from Australia and clustered with some reference isolates of CMV subgroup II from UK (Z12818) and USA (AF127976) in a Neighbour-joining phylogenetic reconstruction. A total of 59 polymorphic (segregating) sites (S) with nucleotide diversity (π) of 0.06218 was evident while no INDEL event was observed in Pakistani isolates. The evolutionary distance of Pakistani CMV isolates was recorded as 0.0657 with each other and 0.0574-0.2964 with other CMV isolates reported elsewhere in the world. A frequent gene flow (Fst = 0.30478 <0.33) was observed between Pakistani and earlier reported CMV isolates. In genetic differentiation analysis, the value of three permutation-based statistical tests viz; Z (84.3011), Snn (0.82456), and Ks* (4.04042) were non-significant. The statistical analysis revealed the values 2.02535, 0.01468, and 0.71862 of Tajima's D, Fu, & Li's F* and D* respectively, demonstrating that the CMV population is under balancing selection.

Resumo Cucumber mosaic cucumovirus (CMV) é uma tremenda ameaça aos vegetais em todo o mundo, inclusive no Paquistão. O presente trabalho foi conduzido para investigar a variabilidade genética de isolados de CMV infectando vegetais de ervilha e espinafre na região de Pothwar, Paquistão. Pesquisas com base em sorologia durante 2016-2017 revelaram 31,70% da incidência geral da doença por CMV em safras de ervilha e espinafre. O ensaio de imunoabsorção enzimática em sanduíche de anticorpo triplo (TAS-ELISA) revelou que todos os isolados positivos pertencem ao subgrupo II do CMV. Dois cDNA selecionados de amostras positivas para ELISA representando cada safra de ervilha e espinafre foram amplificados por PCR (ca.1100 pb) e sequenciados correspondendo ao gene CMV CP que compartilhou 93,7% de identidade de nucleotídeo um com o outro. Ambas as sequências de isolados de ervilha CMV (AAHAP) e espinafre (AARS) do Paquistão foram submetidas ao GenBank como nos de acesso. MH119071 e MH119073, respectivamente. A análise BLAST revelou 93,4% de identidade de sequência do isolado AAHAP com SpK (KC763473) do Irã, enquanto o isolado AARS compartilhou a identidade máxima (94,5%) com a cepa 241 (AJ585519) da Austrália e agrupada com alguns isolados de referência do subgrupo II de CMV do Reino Unido (Z12818) e EUA (AF127976) em uma reconstrução filogenética vizinha. Um total de 59 sítios polimórficos (segregantes) (S) com diversidade de nucleotídeos (π) de 0,06218 foi evidente, enquanto nenhum evento INDEL foi observado em isolados do Paquistão. A distância evolutiva de isolados de CMV do Paquistão foi registrada como 0,0657 entre si e 0,0574-0,2964 com outros isolados de CMV relatados em outras partes do mundo. Um fluxo gênico frequente (Fst = 0,30478 < 0,33) foi observado entre os isolados de CMV do Paquistão e relatados anteriormente. Na análise de diferenciação genética, os valores de três testes estatísticos baseados em permutação viz, Z (84,3011), Snn (0,82456) e Ks * (4,04042) não foram significativos. A análise estatística revelou os valores 2,02535, 0,01468 e 0,71862 de Tajima's D, Fu, & Li's F * e D * respectivamente, demonstrando que a população de CMV está sob seleção de balanceamento.

Cucumber Mosaic Virus-Induced Systemic Necrosis in Arabidopsis thaliana: Determinants and Role in Plant Defense.

Effector-triggered immunity (ETI) is one of the most studied mechanisms of plant resistance to viruses. During ETI, viral proteins are recognized by specific plant R proteins, which most often trigger a hypersensitive response (HR) involving programmed cell death (PCD) and a restriction of infection in the initially infected sites. However, in some plant-virus interactions, ETI leads to a response in which PCD and virus multiplication are not restricted to the entry sites and spread throughout the plant, leading to systemic necrosis. The host and virus genetic determinants, and the consequences of this response in plant-virus coevolution, are still poorly understood. Here, we identified an allelic version of RCY1-an R protein-as the host genetic determinant of broad-spectrum systemic necrosis induced by cucumber mosaic virus (CMV) infection in the Arabidopsis thaliana Co-1 ecotype. Systemic necrosis reduced virus fitness by shortening the infectious period and limiting virus multiplication; thus, this phenotype could be adaptive for the plant population as a defense against CMV. However, the low frequency (less than 1%) of this phenotype in A. thaliana wild populations argues against this hypothesis. These results expand current knowledge on the resistance mechanisms to virus infections associated with ETI in plants.

Development of a multiplex RT-PCR assay for simultaneous detection of Lily symptomless virus, Lily mottle virus, Cucumber mosaic virus, and Plantago asiatica mosaic virus in Lilies.

BACKGROUND: Viral pathogens causing significant economic losses in lilies (Lilium spp. and hybrids) include Lily symptomless virus (LSV), Lily mottle virus (LMoV), Cucumber mosaic virus (CMV), and Plantago asiatica mosaic virus (PlAMV). Rapid and efficient virus detection methods are pivotal to prevent the spread of these viruses. RESULTS: In this study, four specific primer pairs designed from conserved regions of genomic sequences of each virus were used to amplify a 116 bp product for LSV, a 247 bp product for LMoV, a 359 bp product for CMV, and a 525 bp product for PlAMV in a multiplex reverse transcription-polymerase chain reaction (multiplex RT-PCR). The amplified products were clearly separated by 2% agarose gel electrophoresis. The optimal reaction annealing temperature and cycle number were 53.8 °C and 35, respectively. The developed multiplex RT-PCR method was then used to test virus infections from lily samples collected from different regions of China. CONCLUSIONS: An effective multiplex RT-PCR assay was established for the simultaneous detection and differentiation of LSV, LMoV, CMV, and PlAMV in lilies, which offers a useful tool for routine molecular diagnosis and epidemiological studies of these viruses.

Myricetin Derivative LP11 Targets Cucumber Mosaic Virus 2b Protein to Achieve In Vivo Antiviral Activity in Plants.

Cucumber mosaic virus (CMV) 2b protein plays a key role in the process of CMV infecting plants and symptom formation and is a potential molecular target for the control of this important plant virus. The exploitation of antiviral compounds is one of the strategies with the highest input: output ratio in plant protection. In this study, the CMV 2b recombinant protein was cloned, purified, and identified as the target protein by mass spectrometry. Subsequently, we carried out preliminary functional screening of the LP series of myricetin derivatives designed and synthesized in our laboratory and commercial antiviral compounds by microscale thermophoresis (MST), which showed that LP compounds LP4, LP11, LP13, and LP20 interacted well with CMV 2b, with dissociation constant (Kd) values of 1.39, 0.88, 1.52, and 1.77 µM, respectively. Among the commercially available antiviral compounds, ningnanmycin (NNM) was the most active, with a Kd value of 4.09 µM. Then, the strongest binding force to CMV 2b was identified to be from LP11 by isothermal titration calorimetry (ITC) experiments, with a Kd of 1.19 µM. Among the commercial compounds, NNM had the strongest binding force with CMV 2b, with a Kd of 4.62 µM. Through the screening of commercial compounds and LP series compounds by MST and ITC, LP11, NNM (positive control), LP16 (negative control), and the blank control group were selected to test the in vivo impact of LP11 on CMV. Specifically, the screened compounds were sprayed onto CMV-inoculated Nicotiana benthamiana plants to determine their impact on the regulation of CMV pathogenic gene expression, symptoms, and virus titer. The results showed that LP11 had a strong ability to inhibit CMV infection of tobacco at the transcriptional and translational levels. By mutating the CMV 2b protein, the 15th amino acid leucine and the 18th amino acid methionine at the N-terminal region were shown to be potential sites for binding to compound LP11. This finding provided a theoretical basis for screening and developing anti-CMV agents.

Occurrence and Molecular Variability of the Main Kiwifruit Viruses in the Sichuan Province of China.

Viruses cause important yield losses in kiwifruit. Here, we studied the occurrence and population structure of the major kiwifruit viruses in the Sichuan province of China. RT-PCR results showed the presence of Actinidia virus A (AcVA), Actinidia virus B (AcVB), Actinidia chlorotic ringspot-associated virus (AcCRaV), and the cucumber mosaic virus (CMV). AcCRaV was widely distributed, followed by CMV. These two viruses were often detected in co-infection with AcVA and AcVB. The virus detection rate was positively correlated with vine age. Four phylogenetic groups of AcVA and AcVB were identified, with AcVA isolates clustering mainly in subgroup I, and AcVB isolates clustering mainly in subgroups II, III, and IV. All CMV isolates clustered in subgroup II, and AcCRaV isolates clustered in subgroup IA. The genome of AcVA and AcCRaV was under negative selection pressure, while the genome of AcVB and CMV was under positive selection pressure. All the viruses, except AcVB, were in a state of expansion. The full-length genome of the most widely distributed AcCRaV isolate in kiwifruits in the Sichuan province was characterized by sequencing. Unique eight-nucleotide (TTTTTGAT) repeats were found in the 5'-terminal non-coding region of the AcCRaV RNA3 in a possible association with reduced disease symptoms. This is the first study of kiwifruit viruses in Sichuan.