Challenges to Cannabis sativa Production from Pathogens and Microbes-The Role of Molecular Diagnostics and Bioinformatics.

The increased cultivation of Cannabis sativa L. in North America, represented by high Δ9-tetrahydrocannabinol-containing (high-THC) cannabis genotypes and low-THC-containing hemp genotypes, has been impacted by an increasing number of plant pathogens. These include fungi which destroy roots, stems, and leaves, in some cases causing a build-up of populations and mycotoxins in the inflorescences that can negatively impact quality. Viroids and viruses have also increased in prevalence and severity and can reduce plant growth and product quality. Rapid diagnosis of the occurrence and spread of these pathogens is critical. Techniques in the area of molecular diagnostics have been applied to study these pathogens in both cannabis and hemp. These include polymerase chain reaction (PCR)-based technologies, including RT-PCR, multiplex RT-PCR, RT-qPCR, and ddPCR, as well as whole-genome sequencing (NGS) and bioinformatics. In this study, examples of how these technologies have enhanced the rapidity and sensitivity of pathogen diagnosis on cannabis and hemp will be illustrated. These molecular tools have also enabled studies on the diversity and origins of specific pathogens, specifically viruses and viroids, and these will be illustrated. Comparative studies on the genomics and metabolomics of healthy and diseased plants are urgently needed to provide insight into their impact on the quality and composition of cannabis and hemp-derived products. Management of these pathogens will require monitoring of their spread and survival using the appropriate technologies to allow accurate detection, followed by appropriate implementation of disease control measures.

Targeting of cucumber necrosis virus coat protein to the chloroplast stroma attenuates host defense response.

Cucumber necrosis virus (CNV) is a (+)ssRNA virus that elicits spreading local and systemic necrosis in Nicotiana benthamiana. We previously showed that the CNV coat protein (CP) arm functions as a chloroplast transit peptide that targets a CP fragment containing the S and P domains to chloroplasts during infection. Here we show that several CP arm mutants that inefficiently target chloroplasts, along with a mutant that lacks the S and P domains, show an early onset of more localized necrosis along with protracted induction of pathogenesis related protein (PR1a). Agroinfiltrated CNV CP is shown to interfere with CNV p33 and Tomato bushy stunt virus p19 induced necrosis. Additionally, we provide evidence that a CP mutant that does not detectably enter the chloroplast stroma induces relatively higher levels of several plant defense-related genes compared to WT CNV. Together, our data suggest that targeting of CNV CP to the chloroplast stroma interferes with chloroplast-mediated plant defense.

Near-Atomic-Resolution Cryo-Electron Microscopy Structures of Cucumber Leaf Spot Virus and Red Clover Necrotic Mosaic Virus: Evolutionary Divergence at the Icosahedral Three-Fold Axes.

Members of the Tombusviridae family have highly similar structures, and yet there are important differences among them in host, transmission, and capsid stabilities. Viruses in the Tombusviridae family have single-stranded RNA (ssRNA) genomes with T=3 icosahedral protein shells with a maximum diameter of ∼340 Å. Each capsid protein is comprised of three domains: R (RNA binding), S (shell), and P (protruding). Between the R domain and S domain is the "arm" region that studies have shown to play a critical role in assembly. To better understand how the details of structural differences and similarities influence the Tombusviridae viral life cycles, the structures of cucumber leaf spot virus (CLSV; genus Aureusvirus) and red clover necrotic mosaic virus (RCNMV; genus Dianthovirus) were determined to resolutions of 3.2 Å and 2.9 Å, respectively, with cryo-electron microscopy and image reconstruction methods. While the shell domains had homologous structures, the stabilizing interactions at the icosahedral 3-fold axes and the R domains differed greatly. The heterogeneity in the R domains among the members of the Tombusviridae family is likely correlated with differences in the sizes and characteristics of the corresponding genomes. We propose that the changes in the R domain/RNA interactions evolved different arm domain interactions at the ß-annuli. For example, RCNMV has the largest genome and it appears to have created the necessary space in the capsid by evolving the shortest R domain. The resulting loss in RNA/R domain interactions may have been compensated for by increased intersubunit ß-strand interactions at the icosahedral 3-fold axes. Therefore, the R and arm domains may have coevolved to package different genomes within the conserved and rigid shell.IMPORTANCE Members of the Tombusviridae family have nearly identical shells, and yet they package genomes that range from 4.6 kb (monopartite) to 5.3 kb (bipartite) in size. To understand how this genome flexibility occurs within a rigidly conserved shell, we determined the high-resolution cryo-electron microscopy (cryo-EM) structures of cucumber leaf spot virus and red clover necrotic mosaic virus. In response to genomic size differences, it appears that the ssRNA binding (R) domain of the capsid diverged evolutionarily in order to recognize the different genomes. The next region, the "arm," seems to have also coevolved with the R domain to allow particle assembly via interactions at the icosahedral 3-fold axes. In addition, there are differences at the icosahedral 3-fold axes with regard to metal binding that are likely important for transmission and the viral life cycle.

Evidence for the role of basic amino acids in the coat protein arm region of Cucumber necrosis virus in particle assembly and selective encapsidation of viral RNA.

Cucumber necrosis virus (CNV) is a T = 3 icosahedral virus with a (+)ssRNA genome. The N-terminal CNV coat protein arm contains a conserved, highly basic sequence ("KGRKPR"), which we postulate is involved in RNA encapsidation during virion assembly. Seven mutants were constructed by altering the CNV "KGRKPR" sequence; the four basic residues were mutated to alanine individually, in pairs, or in total. Virion accumulation and vRNA encapsidation were significantly reduced in mutants containing two or four substitutions and virion morphology was also affected, where both T = 1 and intermediate-sized particles were produced. Mutants with two or four substitutions encapsidated significantly greater levels of truncated RNA than that of WT, suggesting that basic residues in the "KGRKPR" sequence are important for encapsidation of full-length CNV RNA. Interestingly, "KGRKPR" mutants also encapsidated relatively higher levels of host RNA, suggesting that the "KGRKPR" sequence also contributes to selective encapsidation of CNV RNA.

Encapsidation of Host RNAs by Cucumber Necrosis Virus Coat Protein during both Agroinfiltration and Infection.

UNLABELLED: Next-generation sequence analysis of virus-like particles (VLPs) produced during agroinfiltration of cucumber necrosis virus (CNV) coat protein (CP) and of authentic CNV virions was conducted to assess if host RNAs can be encapsidated by CNV CP. VLPs containing host RNAs were found to be produced during agroinfiltration, accumulating to approximately 1/60 the level that CNV virions accumulated during infection. VLPs contained a variety of host RNA species, including the major rRNAs as well as cytoplasmic, chloroplast, and mitochondrial mRNAs. The most predominant host RNA species encapsidated in VLPs were chloroplast encoded, consistent with the efficient targeting of CNV CP to chloroplasts during agroinfiltration. Interestingly, droplet digital PCR analysis showed that the CNV CP mRNA expressed during agroinfiltration was the most efficiently encapsidated mRNA, suggesting that the CNV CP open reading frame may contain a high-affinity site or sites for CP binding and thus contribute to the specificity of CNV RNA encapsidation. Approximately 0.09% to 0.7% of the RNA derived from authentic CNV virions contained host RNA, with chloroplast RNA again being the most prominent species. This is consistent with our previous finding that a small proportion of CNV CP enters chloroplasts during the infection process and highlights the possibility that chloroplast targeting is a significant aspect of CNV infection. Remarkably, 6 to 8 of the top 10 most efficiently encapsidated nucleus-encoded RNAs in CNV virions correspond to retrotransposon or retrotransposon-like RNA sequences. Thus, CNV could potentially serve as a vehicle for horizontal transmission of retrotransposons to new hosts and thereby significantly influence genome evolution. IMPORTANCE: Viruses predominantly encapsidate their own virus-related RNA species due to the possession of specific sequences and/or structures on viral RNA which serve as high-affinity binding sites for the coat protein. In this study, we show, using next-generation sequence analysis, that CNV also encapsidates host RNA species, which account for ∼0.1% of the RNA packaged in CNV particles. The encapsidated host RNAs predominantly include chloroplast RNAs, reinforcing previous observations that CNV CP enters chloroplasts during infection. Remarkably, the most abundantly encapsidated cytoplasmic mRNAs consisted of retrotransposon-like RNA sequences, similar to findings recently reported for flock house virus (A. Routh, T. Domitrovic, and J. E. Johnson, Proc Natl Acad Sci U S A 109:1907-1912, 2012). Encapsidation of retrotransposon sequences may contribute to their horizontal transmission should CNV virions carrying retrotransposons infect a new host. Such an event could lead to large-scale genomic changes in a naive plant host, thus facilitating host evolutionary novelty.

The Cucumber leaf spot virus p25 auxiliary replicase protein binds and modifies the endoplasmic reticulum via N-terminal transmembrane domains.

Cucumber leaf spot virus (CLSV) is a member of the Aureusvirus genus, family Tombusviridae. The auxiliary replicase of Tombusvirids has been found to localize to endoplasmic reticulum (ER), peroxisomes or mitochondria; however, localization of the auxiliary replicase of aureusviruses has not been determined. We have found that the auxiliary replicase of CLSV (p25) fused to GFP colocalizes with ER and that three predicted transmembrane domains (TMDs) at the N-terminus of p25 are sufficient for targeting, although the second and third TMDs play the most prominent roles. Confocal analysis of CLSV infected 16C plants shows that the ER becomes modified including the formation of punctae at connections between ER tubules and in association with the nucleus. Ultrastructural analysis shows that the cytoplasm contains numerous vesicles which are also found between the perinuclear ER and nuclear membrane. It is proposed that these vesicles correspond to modified ER used as sites for CLSV replication.

The p33 auxiliary replicase protein of Cucumber necrosis virus targets peroxisomes and infection induces de novo peroxisome formation from the endoplasmic reticulum.

Tombusviruses replicate on pre-existing organelles such as peroxisomes or mitochondria, the membranes of which become extensively reorganized into multivesicular bodies (MVBs) during the infection process. Cucumber necrosis virus (CNV) has previously been shown to replicate in association with peroxisomes in yeast. We show that CNV induces MVBs from peroxisomes in infected plants and that GFP-tagged p33 auxiliary replicase protein colocalizes with YFP(SKL), a peroxisomal marker. Most remarkably, the ER of CNV infected Nicotiana benthamiana 16C plants undergoes a dramatic reorganization producing numerous new peroxisome-like structures that associate with CNV p33, thus likely serving as a new site for viral RNA replication. We also show that plants agroinfiltrated with p33 develop CNV-like necrotic symptoms which are associated with increased levels of peroxide. Since peroxisomes are a site for peroxide catabolism, and peroxide is known to induce plant defense responses, we suggest that dysfunctional peroxisomes contribute to CNV induced necrosis.

Cucumber necrosis virus p20 is a viral suppressor of RNA silencing.

The p20 protein encoded by the tombusvirus, Cucumber necrosis virus has previously been shown to be involved in host pathogenicity and shares sequence similarity with the Tomato bushy stunt virus p19 suppressor of silencing. Using a virus-induced gene silencing (VIGS) assay, we show that p20 is a viral suppressor of RNA silencing (VSR) in infected plants. In addition, a CNV p20-knockout mutant showed a decline in viral RNA accumulation in infected plants, consistent with the role of p20 in suppression of RNA silencing. However, unexpectedly, all GFP transgenic plants co-infiltrated with p20 and GFP displayed RNA silencing using an Agrobacterium-mediated silencing assay. Detailed RNA analysis of GFP mRNA levels in p20 agro-infiltrated plants revealed that p20 did initially display suppressor activity but this was rapidly overcome by RNA silencing. p20 expression levels in agro-infiltrated plants were shown to be approximately 50-fold lower than that of the TBSV p19 silencing suppressor, consistent with the notion that p20 dosage levels are not sufficient to suppress RNA silencing in the Agrobacterium-mediated system. Our results suggest that a viral-based VIGS assay may be required for identifying VSRs encoded by some plant viruses. Based on bioinformatics studies the mechanism of suppression of silencing by p20 is predicted to be similar to that of the TBSV p19 suppressor.

A highly basic KGKKGK sequence in the RNA-binding domain of the Cucumber necrosis virus coat protein is associated with encapsidation of full-length CNV RNA during infection.

The Cucumber necrosis virus particle is a T=3 icosahedron consisting of 180 identical coat protein (CP) subunits. The N-terminal 58 aa residue segment of the CP R domain is believed to bind viral RNA within virions and during assembly. We report results of in vivo experiments that examine the role of the R domain in assembly. Deletion analyses identified 3 conserved 5-10 aa regions as playing critical roles. A highly basic KGKKGK sequence was found to be both necessary and sufficient for encapsidation of the full-length genome and polymorphic virions were produced in mutants lacking the KGKKGK sequence. The amount of full-length RNA present in virions was substantially reduced in R domain mutants where 2 of the 4 lysine residues were substituted with alanine, whereas substitution of 4 lysines by arginine had only a modest effect. The potential role of the R domain in formation of a scaffold for particle assembly is discussed.

Induction of particle polymorphism by cucumber necrosis virus coat protein mutants in vivo.

The Cucumber necrosis virus (CNV) particle is a T=3 icosahedron consisting of 180 identical coat protein (CP) subunits. Plants infected with wild-type CNV accumulate a high number of T=3 particles, but other particle forms have not been observed. Particle polymorphism in several T=3 icosahedral viruses has been observed in vitro following the removal of an extended N-terminal region of the CP subunit. In the case of CNV, we have recently described the structure of T=1 particles that accumulate in planta during infection by a CNV mutant (R1+2) in which a large portion of the N-terminal RNA binding domain (R-domain) has been deleted. In this report we further describe properties of this mutant and other CP mutants that produce polymorphic particles. The T=1 particles produced by R1+2 mutants were found to encapsidate a 1.9-kb RNA species as well as smaller RNA species that are similar to previously described CNV defective interfering RNAs. Other R-domain mutants were found to encapsidate a range of specifically sized less-than-full-length CNV RNAs. Mutation of a conserved proline residue in the arm domain near its junction with the shell domain also influenced T=1 particle formation. The proportion of polymorphic particles increased when the mutation was incorporated into R-domain deletion mutants. Our results suggest that both the R-domain and the arm play important roles in the formation of T=3 particles. In addition, the encapsidation of specific CNV RNA species by individual mutants indicates that the R-domain plays a role in the nature of CNV RNA encapsidated in particles.

Structures of T=1 and T=3 particles of cucumber necrosis virus: evidence of internal scaffolding.

Cucumber necrosis virus (CNV) is a member of the genus Tombusvirus, of which tomato bushy stunt virus (TBSV) is the type member. The capsid protein for this group of viruses is composed of three major domains: the R domain, which interacts with the RNA genome: the S domain, which forms the tight capsid shell: and the protruding P domain, which extends approximately 40 Angstrom from the surface. Here, we present the cryo-transmission electron microscopy structures of both the T=1 and T=3 capsids to a resolution of approximately 12 Angstrom. The T=3 capsid is essentially identical with that of TBSV, and the T=1 particles are well described by the A subunit pentons from TBSV. Perhaps most notable is the fact that the T=3 particles have an articulated internal structure with two major internal shells, while the internal core of the T=1 particle is essentially disordered. These internal shells of the T=3 capsid agree extremely well in both dimension and character with published neutron-scattering results. This structure, combined with mutagenesis results in the accompanying article, suggests that the R domain forms an internal icosahedral scaffold that may play a role in T=3 capsid assembly. In addition, the N-terminal region has been shown to be involved in chloroplast targeting. Therefore, this region apparently has remarkably diverse functions that may be distributed unevenly among the quasi-equivalent A, B, and C subunits.

A 38-amino-acid sequence encompassing the arm domain of the cucumber necrosis virus coat protein functions as a chloroplast transit Peptide in infected plants.

Experiments to determine the subcellular location of the coat protein (CP) of the tombusvirus Cucumber necrosis virus (CNV) have been conducted. By confocal microscopy, it was found that an agroinfiltrated CNV CP-green fluorescent protein (GFP) fusion targets chloroplasts in Nicotiana benthamiana leaves and that a 38-amino-acid (aa) region that includes the complete CP arm region plus the first 4 amino acids of the shell domain are sufficient for targeting. Western blot analyses of purified and fractionated chloroplasts showed that the 38-aa region directs import to the chloroplast stroma, suggesting that the CNV arm can function as a chloroplast transit peptide (TP) in plants. Several features of the 38-aa region are similar to features typical of chloroplast TPs, including (i) the presence of an alanine-rich uncharged region near the N terminus, followed by a short region rich in basic amino acids; (ii) a conserved chloroplast TP phosphorylation motif; (iii) the requirement that the CNV 38-aa sequence be present at the amino terminus of the imported protein; and (iv) specific proteolytic cleavage upon import into the chloroplast stroma. In addition, a region just downstream of the 38-aa sequence contains a 14-3-3 binding motif, suggesting that chloroplast targeting requires 14-3-3 binding, as has been suggested for cellular proteins that are targeted to chloroplasts. Chloroplasts of CNV-infected plants were found to contain CNV CP, but only the shell and protruding domain regions were present, indicating that CNV CP enters chloroplasts during infection and that proteolytic cleavage occurs as predicted from agroinfiltration studies. We also found that particles of a CNV CP mutant deficient in externalization of the arm region have a reduced ability to establish infection. The potential biological significance of these findings is discussed.

Molecular aspects of plant virus transmission by olpidium and plasmodiophorid vectors.

The genome structures of a large number of viruses transmitted by olpidium and plasmodiophorid vectors have been determined. The viruses are highly diverse, belonging to 12 genera in at least 4 families. Plasmodiophorids are now classified as protists rather than true fungi. This finding, along with the recognition of the great variety of viruses transmitted by olpidium and plasmodiophorid vectors, will likely lead to an elaboration of the details of in vitro and in vivo transmission mechanisms. Recent progress in elucidating the interaction between Cucumber necrosis virus (CNV) and its zoospore vector suggests that specific sites on the capsid as well as on the zoospore are involved in transmission. Moreover, some features of CNV/zoospore attachment are similar to poliovirus/host cell interactions, suggesting evolutionary conservation of functional features of plant and animal virus capsids.

Evidence that vector transmission of a plant virus requires conformational change in virus particles.

Transmission of Cucumber necrosis virus (CNV) by zoospores of its fungal vector, Olpidium bornovanus, involves specific adsorption of virus particles onto the zoospore plasmalemma prior to infestation of cucumber roots by virus-bound zoospores. Previous work has shown that specific components of both CNV and zoospores are required for successful CNV/zoospore recognition. Here, we show that limited trypsin digestion of CNV following in vitro CNV/zoospore binding assays, results in the production of specific proteolytic digestion products under conditions where native CNV is resistant. The proteolytic digestion pattern of zoospore-bound CNV was found to be similar to that of swollen CNV particles produced in vitro, suggesting that zoospore-bound CNV is in an altered conformational state, perhaps similar to that of swollen CNV. We show that an engineered CNV mutant (Pro73Gly) in which a conserved proline residue (Pro73) in the beta-annulus of the CP arm is changed to glycine is resistant to proteolysis following in vitro zoospore binding assays. Moreover, Pro73Gly particles are transmitted only poorly by O.bornovanus. Together, the results of these studies suggest that CNV undergoes conformational change upon zoospore binding and that the conformational change is important for CNV transmissibility.