Harmful viruses on stone fruit crops and sanitation methods

Relevance. Viral diseases can significantly reduce the yield of stone fruit crops. More than 30 viruses have been characterized on stone fruits crops, among which the most harmful are Plum pox virus (PPV), Prunus dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), Cherry leaf roll spot virus (CLRV), Apple chlorotic leaf spot virus (ACLSV). Viral diseases monitoring is essential for controlling the viruses prevalence and choosing a control strategy. In the absence of healthy plants of a certain variety, health improvement is carried out using laboratory methods, including thermotherapy. Sanitation methods need to be improved in relation to the culture characteristics and the virus type. Of considerable interest is the development of techniques that reduce the viruses concentration when growing stone fruit trees in the field.Methods. During 2016–2020 using the ELISA (“Loewe” diagnostic kits) diagnostics of viruses on varieties and clonal rootstocks of cherry, sweet cherry and plum (660 plants) was carried out in the conditions of the Moscow region. For plant health in 2019–2021 used thermotherapy for 3 months. To study the effect of Pharmayod (“Farmbiomed”) on viruses in open ground, 24 plum plants of 5 varieties were treated with this drug at a concentration of 0.3 ml/l.Results. The total prevalence of viruses on cherries varieties was 44%, sweet cherries — 40%, plums — 59%, on clonal rootstocks — 46, 55 and 56% respectively. The highest incidence of PNRSV and PDV viruses has been established. Cherry plants of 11 varieties, free from the main harmful viruses, 4 varieties of sweet cherries, 12 varieties of plums and 9 forms of clonal rootstocks were revealed. The use of the Pharmayod on plum trees in the open field contributed to a decrease in the infection index of the studied viruses. Plum plants in a heat chamber were characterized by a higher survival rate and growth parameters in comparison with cherry and sweet cherry. After the completion of thermotherapy, a significant decrease in the index of infection in plants was noted.

N6-Methyladenosine Regulatory Machinery is Involved In Viral Infection of Plum Pox Virus And Potato Virus Y In Nicotiana Benthamiana

Abstract N6-methyladenosine (m6A) is a post-transcriptional modification of biological mRNA and non-coding RNAs, which by regulating the mRNA stability and translation. It has been demonstrated that m6A methylation has a regulatory effect on human RNA virus replication. In this project, Plum pox virus (PPV) and Potato Y virus (PVY) were used to examine the m6A modification in Nicotiana benthamiana during natural infection. The results showed that the global level of m6A in both PVY and PPV infected plants were significantly decreased than non-infected plants. Particularly, the PPV and PVY infection could alter the m6A level of the host endogenous gene. This is suggesting that plant viruses may disrupt the balance of the m6A in plant. Meanwhile, we found that viral genome RNA can be targeted by m6A methylation. Two m6A-enrich regions in PPV genome RNA and four in PVY genome RNA were detected, which are located in the coding region of viruses. Based on the ALKB and METTL sequences in the transcriptome sequencing data of the virus-infected plant, we cloned 2 NbALKB genes and 2 NbMETTL genes in N. benthamiana . According to results of transient expression and VIGS assay, NbALKB gene appears slightly contributing PPV and PVY infection. NbMETTL gene showed certain inhibition effect in PPV infection, but not PVY. Therefore, our data suggested that m6A methylation in plant might be an anti-viral strategy in some plant viruses.

An Importin-β-like Protein from Nicotiana benthamiana Interacts with the RNA Silencing Suppressor P1b of the Cucumber Vein Yellowing Virus, Modulating Its Activity

During a plant viral infection, host–pathogen interactions are critical for successful replication and propagation of the virus through the plant. RNA silencing suppressors (RSSs) are key players of this interplay, and they often interact with different host proteins, developing multiple functions. In the Potyviridae family, viruses produce two main RSSs, HCPro and type B P1 proteins. We focused our efforts on the less known P1b of cucumber vein yellowing virus (CVYV), a type B P1 protein, to try to identify possible factors that could play a relevant role during viral infection. We used a chimeric expression system based on plum pox virus (PPV) encoding a tagged CVYV P1b in place of the canonical HCPro. We used that tag to purify P1b in Nicotiana-benthamiana-infected plants and identified by mass spectrometry an importin-β-like protein similar to importin 7 of Arabidopsis thaliana. We further confirmed the interaction by bimolecular fluorescence complementation assays and defined its nuclear localization in the cell. Further analyses showed a possible role of this N. benthamiana homolog of Importin 7 as a modulator of the RNA silencing suppression activity of P1b.

Proteolytic processing of plant proteins by potyvirus NIa proteases

The NIa protease of potyviruses is a chymotrypsin-like cysteine protease related to the picornavirus 3C protease. It is also a multifunctional protein known to play multiple roles during virus infection. Picornavirus 3C proteases cleave hundreds of host proteins to facilitate virus infection. However, whether or not potyvirus NIa proteases cleave plant proteins has so far not been tested. Regular expression search using the cleavage site consensus sequence [EQN]xVxH[QE]/[SGTA] for the plum pox virus (PPV) protease identified 90-94 putative cleavage events in the proteomes of Prunus persica (a crop severely affected by PPV), Arabidopsis thaliana and Nicotiana benthamiana (two experimental hosts). In vitro processing assays confirmed cleavage of six A. thaliana and five P. persica proteins by the PPV protease. These proteins were also cleaved in vitro by the protease of turnip mosaic virus (TuMV), which has a similar specificity. We confirmed in vivo cleavage of a transiently expressed tagged version of AtEML2, an EMSY-like protein belonging to a family of nuclear histone readers known to be involved in pathogen resistance. Cleavage of AtEML2 was efficient and was observed in plants that co-expressed the PPV or TuMV NIa proteases or in plants that were infected with TuMV. We also show partial in vivo cleavage of AtDUF707, a membrane protein annotated as lysine ketoglutarate reductase trans-splicing protein. Although cleavage of the corresponding endogenous plant proteins remains to be confirmed, the results show that a plant virus protease can cleave host proteins during virus infection and highlight a new layer of plant-virus interactions. Importance Viruses are highly adaptive and use multiple molecular mechanisms to highjack or modify the cellular resources to their advantage. They must also counteract or evade host defense responses. One well-characterized mechanism used by vertebrate viruses is the proteolytic cleavage of host proteins to inhibit the activities of these proteins and/or to produce cleaved protein fragments that are beneficial to the virus infection cycle. Even though almost half of the known plant viruses encode at least one protease, it was not known whether plant viruses employ this strategy. Using an in silico prediction approach and the well-characterized specificity of potyvirus NIa proteases, we were able to identify hundreds of putative cleavage sites in plant proteins, several of which were validated by downstream experiments. It can be anticipated that many other plant virus proteases also cleave host proteins and that the identification of these cleavage events will lead to novel antiviral strategies.

Identification and Characterization of a Novel Potyvirus Infecting Paris Yunnanensis

The complete genomic sequence of a novel potyvirus from Paris yunnanensis was determined by high-throughput sequencing then confirmed by Sanger sequencing. Its genomic RNA consists 9600 nucleotides (nt) excluding the 3’-terminal poly (A) tail, containing a typical large open reading frame (ORF) of potyviruses and encoding a putative polyprotein of 3098 amino acids (aa). Pairwise comparison analysis showed the virus shares sequence identity with other members of Potyvirus was 53.0% to 57.8% at genome sequence level, and 39.3% to 51.2% at polyprotein sequence level. Phylogenetic analysis indicated that the virus was most closely related to the subgroup of plum pox virus and that of chilli veinal mottle virus within the genus Potyvirus. These results suggest that the virus should be considered as a distinct species within the genus Potyvirus and was tentatively named as “Paris mottle associated virus” (PMaV).

Long-Term Efficacy and Safety of RNAi-Mediated Virus Resistance in ‘HoneySweet’ Plum

Interfering RNA technology has been established as an effective strategy to protect plants against viral infection. Despite this success, interfering RNA (RNAi) has rarely been applied due to the regulatory barriers that confront genetically engineered plants and concerns over possible environmental and health risks posed by non-endogenous small RNAs. ‘HoneySweet’ was developed as a virus-resistant plum variety that is protected by an RNAi-mediated process against Sharka disease caused by the plum pox virus. ‘HoneySweet’ has been approved for cultivation in the United States but not in countries where the plum pox virus is endemic. In this study, we evaluated the long-term efficacy of virus resistance in ‘HoneySweet,’ the nature and stability of its sRNA profile, and the potential health risks of consuming ‘HoneySweet’ plums. Graft-challenged ‘HoneySweet’ trees carrying large non-transgenic infected limbs remained virus-free after more than 10 years in the field, and the viral sequences from the non-transgenic infected limbs showed no evidence of adaptation to the RNAi-based resistance. Small RNA profiling revealed that transgene-derived sRNA levels were stable across different environments and, on average, were more than 10 times lower than those present in symptom-less fruits from virus-infected trees. Comprehensive 90-day mouse feeding studies showed no adverse health impacts in mice, and there was no evidence for potential siRNA off-target pathologies predicted by comparisons of the most abundant transgene-derived sRNAs to the mouse genome. Collectively, the data confirmed that RNAi provides a highly effective, stable, and safe strategy to combat virus diseases in crop plants.

Viral reservoir capacity of wild Prunus alternative hosts of plum pox virus through multiple cycles of transmission and dormancy

Plum pox virus (PPV) is a significant pathogen of Prunus worldwide and is known for having a broad experimental host range. Many of these hosts represent epidemiological risks as potential wild viral reservoirs. A comparative study of the PPV reservoir capacity of three commonly found native North American species, western choke cherry (P. virginiana var. demissa), black cherry (P. serotina), and American plum (P. americana) was conducted. Pennsylvania isolates of PPV-D were transmitted from the original host peach (P. persica cv. GF305) to all three species. Viral accumulation and transmission rates to alternative hosts and peach were monitored over the course of five vegetative growth and cold induced dormancy (CID) cycles. The three alternative host species demonstrated differences in their ability to maintain PPV-D and the likelihood of transmission to additional alternative hosts or back transmission to peach. Western choke cherry had low (5.8%) initial infection levels, PPV-D was not transmissible to additional western choke cherry, and transmission of PPV-D from western choke cherry to peach was only possible before the first CID cycle. Black cherry had intermediate initial infection levels (26.6%) but did not maintain high infection levels after repeated CID cycles. Conversely, American plum had a high level (50%) of initial infection that was not significantly different from initial infection in peach (72.2%) and maintained moderate levels (15-25%) of infection and PPV-D transmission to both American plum and peach through all five cycles of CID. Our results indicate that American plum has the greatest potential to act as a reservoir host for Pennsylvania isolates of PPV-D.

Deciphering Prunus Responses to PPV Infection: A Way toward the Use of Metabolomics Approach for the Diagnostic of Sharka Disease

Sharka disease, caused by Plum pox virus (PPV), induces several changes in Prunus. In leaf tissues, the infection may cause oxidative stress and disrupt the photosynthetic process. Moreover, several defense responses can be activated after PPV infection and have been detected at the phytohormonal, transcriptomic, proteomic, and even translatome levels. As proposed in this review, some responses may be systemic and earlier to the onset of symptoms. Nevertheless, these changes are highly dependent among species, variety, sensitivity, and tissue type. In the case of fruit tissues, PPV infection can modify the ripening process, induced by an alteration of the primary metabolism, including sugars and organic acids, and secondary metabolism, including phenolic compounds. Interestingly, metabolomics is an emerging tool to better understand Prunus–PPV interactions mainly in primary and secondary metabolisms. Moreover, through untargeted metabolomics analyses, specific and early candidate biomarkers of PPV infection can be detected. Nevertheless, these candidate biomarkers need to be validated before being selected for a diagnostic or prognosis by targeted analyses. The development of a new method for early detection of PPV-infected trees would be crucial for better management of the outbreak, especially since there is no curative treatment.