Citrus sudden death (CSD) has caused the death of approximately four million orange trees in a very important citrus region in Brazil. Although its etiology is still not completely clear, symptoms and distribution of affected plants indicate a viral disease. In a search for viruses associated with CSD, we have performed a comparative high-throughput sequencing analysis of the transcriptome and small RNAs from CSD-symptomatic and -asymptomatic plants using the Illumina platform. The data revealed mixed infections that included Citrus tristeza virus (CTV) as the most predominant virus, followed by the Citrus sudden death-associated virus (CSDaV), Citrus endogenous pararetrovirus (CitPRV) and two putative novel viruses tentatively named Citrus jingmen-like virus (CJLV), and Citrus virga-like virus (CVLV). The deep sequencing analyses were sensitive enough to differentiate two genotypes of both viruses previously associated with CSD-affected plants: CTV and CSDaV. Our data also showed a putative association of the CSD-symptomatic plants with a specific CSDaV genotype and a likely association with CitPRV as well, whereas the two putative novel viruses showed to be more associated with CSD-asymptomatic plants. This is the first high-throughput sequencing-based study of the viral sequences present in CSD-affected citrus plants, and generated valuable information for further CSD studies.

Plant viruses are transmitted mechanically or by vegetative propagation, and by vectors such as arthropods, fungi, nematodes, or parasitic plants. Sources to access available information regarding plant virus transmissions are scattered and require extensive literature searches. Here, a recently created plant virus transmission database is described. This was developed to provide access to the modes of transmission and vectors of over 1600 plant viruses. The database was compiled using over 3500 publication records spanning the last 100 years. The information is publicly accessible via https://library.wur.nl/WebQuery/virus and fully searchable by virus name, taxonomic position, mode of transmission or vector.

Due to their high specificity and efficacy, RNA interference (RNAi)-based strategies have been used for fundamental functional genomics studies in a number of insects. However, its potential for translational applications in pest management is also of great interest. The lack of suitable RNAi triggering approaches, however, so far has largely precluded the implementation of RNAi-based approaches to target aphids. In this work, we first demonstrate that Flock House virus (FHV), an insect virus, can infect multiple aphid species, including the green peach aphid, Myzus persicae (M. persicae), the corn leaf aphid, Rhopalosiphum maidis (R. maidis), and the bird cherry-oat aphid, Rhopalosiphum padi (R. padi), by both microinjection and oral feeding. Using green fluorescent protein (GFP) as an indicator, we showed that the defective interfering RNA (DI-634) of FHV RNA2, which is generated autonomously during wild-type (WT) virus replication, can carry foreign sequences, and further for their functional expression. More importantly, the engineered DI-634 was incorporated into virus particles in co-infections with WT FHV. Using FHV virions containing genetically modified DI-634, the accumulation levels of the M. persicae mRNAs for Cathepsin L (CatL) and Sugar Transporter 4 (ST4), were decreased by ~ 35% and ~ 30–50%, respectively when virions were injected intrathoracically into aphids. Finally, and of more practical relevance, oral acquisition of these engineered FHV virions caused lethality of M. persicae. In summary, as a proof-of-concept, our work demonstrates that FHV can be a valuable RNAi tool for fundamental research, and suggests opportunities for using engineered insect viruses as biological agents for aphid pest control.

Citrus sudden death-associated virus (CSDaV) is a monopartite positive-sense single-stranded RNA virus that was suggested to be associated with citrus sudden death (CSD) disease in Brazil. Here, we report the first study of the genetic structure and molecular variability among 31 CSDaV isolates collected from both symptomatic and asymptomatic trees in CSD-affected areas. Analyses of partial nucleotide sequences of five domains of the CSDaV genomic RNA, including those encoding for the methyltransferase, the multi-domain region (MDR), the helicase, the RNA-dependent RNA polymerase and the coat protein, showed that the MDR coding region was the most diverse region assessed here, and a possible association between this region and virus adaption to different host or plant tissues is considered. Overall, the nucleotide diversity (π) was low for CSDaV isolates, but the phylogenetic analyses revealed the predominance of two main groups, one of which showed a higher association with CSD-symptomatic plants. Isolates obtained from CSD-symptomatic plants, compared to those obtained from asymptomatic plants, showed higher nucleotide diversity, nonsynonymous and synonymous substitution rates and number of amino acid changes on the coding regions located closer to the 5’ end region of the genomic RNA. This work provides new insights into the genetic diversity of the CSDaV, giving support for further epidemiological studies.

Small RNAs (sRNAs) are the hallmark and main effectors of RNA silencing and therefore are involved in major biological processes in plants, such as regulation of gene expression, antiviral defense, and plant genome integrity. The mechanisms of sRNA amplification as well as their mobile nature and rapid generation suggest sRNAs as potential key modulators of intercellular and interspecies communication in plant-pathogen–pest interactions. Plant endogenous sRNAs can act in cis to regulate plant innate immunity against pathogens, or in trans to silence pathogens’ messenger RNAs (mRNAs) and impair virulence. Likewise, pathogen-derived sRNAs can act in cis to regulate expression of their own genes and increase virulence towards a plant host, or in trans to silence plant mRNAs and interfere with host defense. In plant viral diseases, virus infection alters the composition and abundance of sRNAs in plant cells, not only by triggering and interfering with the plant RNA silencing antiviral response, which accumulates virus-derived small interfering RNAs (vsiRNAs), but also by modulating plant endogenous sRNAs. Here, we review the current knowledge on the nature and activity of virus-responsive sRNAs during virus–plant interactions and discuss their role in trans-kingdom modulation of virus vectors for the benefit of virus dissemination.

• Plant viruses can be engineered as expression vectors of heterologous proteins (hp). • New expression vectors were built based on citrus sudden death-associated virus (CSDaV). • Hp is highly expressed when replacing the major CSDaV coat protein (CPp21). • Hp can be incorporated into CSDaV (chimeric) virions when fused to the coat protein. • Chimeric virus-like particles are produced by in planta co-expression of hp-CPp21 and CPp21.