Lettuce Infectious Yellows Virus Research Articles

Lettuce infectious yellows virus (infectious yellows of lettuce)

Lettuce infectious yellows virus (infectious yellows of lettuce)

Plasticity of the lettuce infectious yellows virus minor coat protein (CPm) in mediating the foregut retention and transmission of a chimeric CPm mutant by whitefly vectors.

Transmission of the crinivirus, lettuce infectious yellows virus (LIYV), is determined by a minor coat protein (CPm)-mediated virion retention mechanism located in the foregut of its whitefly vector. To better understand the functions of LIYV CPm, chimeric CPm mutants engineered with different lengths of the LIYV CPm amino acid sequence and that of the crinivirus, lettuce chlorosis virus (LCV), were constructed based on bioinformatics and sequence alignment data. The 485 amino acid-long chimeric CPm of LIYV mutant, CPmP-1, contains 60 % (from position 3 to 294) of LCV CPm amino acids. The chimeric CPm of mutants CPmP-2, CPmP-3 and CPmP-4 contains 46 (position 3 to 208), 51 (position 3 to 238) and 41 % (position 261 to 442) of LCV CPm amino acids, respectively. All four mutants moved systemically, expressed the chimeric CPm and formed virus particles. However, following acquisition feeding of the virus preparations, only CPmP-1 was retained in the foreguts of a significant number of vectors and transmitted. In immuno-gold labelling transmission electron microscopy (IGL-TEM) analysis, CPmP-1 particles were distinctly labelled by antibodies directed against the LCV but not LIYV CPm. In contrast, CPmP-4 particles were not labelled by antibodies directed against the LCV or LIYV CPm, while CPmP-2 and -3 particles were weakly labelled by anti-LIYV CPm but not anti-LCV CPm antibodies. The unique antibody recognition and binding pattern of CPmP-1 was also displayed in the foreguts of whitefly vectors that fed on CPmP-1 virions. These results are consistent with the hypothesis that the chimeric CPm of CPmP-1 is incorporated into functional virions, with the LCV CPm region being potentially exposed on the surface and accessible to anti-LCV CPm antibodies.

Nuances of Whitefly Vector-Crinivirus Interactions Revealed in the Foregut Retention and Transmission of Lettuce Chlorosis Virus by Two Bemisia tabaci Cryptic Species.

Lettuce infectious yellows virus is the first crinivirus for which the retention of purified virions ingested into the whitefly (Bemisia tabaci New World (NW)) vector’s foregut, has been demonstrated to be a requisite for successful virus transmission. This key finding supports the hypothesis that the determinant of foregut retention and transmission is present on the virion itself. However, whether this is also true for other criniviruses has not been established. Here, we provide evidence that lettuce chlorosis virus (LCV) acquired from plants is retained in the foreguts of both the B. tabaci NW and Middle East–Asia Minor 1 (MEAM1) vector species and transmitted upon inoculation feeding. An association between foregut retention and transmission by NW vectors is also observed following the acquisition and inoculation feeding of LCV virions purified using a standard procedure involving 2% or 4% (v/v) Triton™ X-100 (TX-100). However, while virions purified with 2% or 4% TX-100 are also retained in the foreguts of MEAM1 vectors, transmission is observed with the 4% TX-100-purified virions or when more vectors are used for acquisition and inoculation feeding. These results suggest that an intrinsic difference exists between NW and MEAM1 vectors in their interactions with, and transmission of, LCV virions.

First Report of Cucurbit chlorotic yellows virus infecting Cucumber Plants in Spain.

During the winter 2018, symptoms of leaf chlorotic spots (Figure 1) followed by symptoms of leaf interveinal chlorosis (Figure 2) and severe chlorosis in basal leaves were observed in cucumber cv Laredo (Cucumis sativus) plants in three separated greenhouses, sited in distinct locations in southern Spain. In all cases, Bemisia tabaci populations were observed on infected plants. The symptomology observed was similar to that caused by whitefly transmitted Cucurbit yellow stunting disorder virus (CYSDV, genus Crinivirus, family Closteroviridae), which is usually found infecting cucumber plants in this geographical area (1). Samples from four different cucumber plants of distinct greenhouses were collected and tested for the presence of CYSDV. Total RNA was extracted from the samples using the NucleoSpin RNA Plant kit (Macherey-Nagel, Germany). Molecular detection of CYSDV was performed using the multiplex and degenerate primer RT-PCR method (2), specific to the region of the highly conserved RNA-dependent RNA polymerase (RdRp) gene of criniviruses, which also detects other criniviruses such as Lettuce infectious yellows virus (LIYV) and Beet pseudo-yellows virus (BPYV). Results indicated that the viral species CYSDV, LIYV and BPYV were not detected in the four cucurbit plant samples. In 2004, an emergent crinivirus (Cucurbit chlorotic yellows virus, CCYV), inducing symptoms similar to those caused by CYSDV, was described infecting cucurbits in Japan (3). Recently, CCYV was detected in 2011 in Greece (4) and in 2014 in Egypt (5) and Saudi Arabia (6). Therefore, the four RNA samples were tested for the presence of the CCYV by a RT-PCR method previously described (7). Specific primers were designed to amplify 336 nt of the capsid protein (CP) gene and 680 nt of the RdRp gene, located on CCYV genomic RNA 1 and RNA 2, respectively. In all cases, clear cDNA bands of both expected sizes were detected for each cucumber sample that were then purified and sequenced via Sanger technology. BLAST analysis of those sequences showed 99% identity with the nucleotide sequence of the CP and RpRd genes from the CCYV isolates from Greece (LT992911, LT992910), China (KY400633.1, KX118632) and Taiwan (JF502222). To our knowledge, this is the first report of CCYV infecting cucurbits in Spain. Probably CCYV has been spread throughout the Mediterranean basin, remaining undetected due to the yellowing symptom similarities between CYSDV and CCYV. Detection of the emergent virus CCYV in Spain represents a new threat for the horticultural area of southern Europe.

A Distinct, Non-Virion Plant Virus Movement Protein Encoded by a Crinivirus Essential for Systemic Infection

Plant-infecting viruses utilize various strategies involving multiple viral and host factors to achieve successful systemic infections of their compatible hosts. Lettuce infectious yellows virus (LIYV), genus Crinivirus, family Closteroviridae, has long, filamentous flexuous virions and causes phloem-limited infections in its plant hosts. The LIYV-encoded P26 is a distinct non-virion protein that shows no similarities to proteins in current databases: it induces plasmalemma deposits over plasmadesmata (PD) pit fields and is speculated to have roles in LIYV virion transport within infected plants. In this study, P26 was demonstrated to be a PD-localized protein, and its biological significance was tested in planta by mutagenesis analysis. An LIYV P26 knockout mutant (P26X) showed viral RNA replication and virion formation in inoculated leaves of Nicotiana benthamiana plants, but failed to give systemic infection. Confirmation by using a modified green fluorescent protein (GFP)-tagged LIYV P26X showed GFP accumulation only in infiltrated leaf tissues, while wild-type LIYV GFP readily spread systemically in the phloem. Attempts to rescue P26X by complementation in trans were negative. However a translocated LIYV P26 gene in the LIYV genome rescued systemic infection, but P26 orthologs from other criniviruses did not. Mutagenesis in planta assays showed that deletions in P26, as well as 2 of 11 specific alanine-scanning mutants, abolished the ability to systemically infect N. benthamiana IMPORTANCE Plant viruses encode specific proteins that facilitate their ability to establish multicellular/systemic infections in their host plants. Relatively little is known of the transport mechanisms for plant viruses whose infections are phloem limited, including those of the family Closteroviridae. These viruses have complex, long filamentous virions that spread through the phloem. Lettuce infectious yellows virus (LIYV) encodes a non-virion protein, P26, which forms plasmalemma deposits over plasmodesmata pit fields, and LIYV virions are consistently found attached to those deposits. Here we demonstrate that P26 is a unique movement protein required for LIYV systemic infection in plants. LIYV P26 shows no sequence similarities to other proteins, but other criniviruses encode P26 orthologs. However, these failed to complement movement of LIYV P26 mutants.

Two Crinivirus-Conserved Small Proteins, P5 and P9, Are Indispensable for Efficient Lettuce infectious yellows virus Infectivity in Plants.

Genomic analysis of Lettuce infectious yellows virus (LIYV) has revealed two short open reading frames (ORFs) on LIYV RNA2, that are predicted to encode a 5-kDa (P5) and a 9-kDa (P9) protein. The P5 ORF is part of the conserved quintuple gene block in the family Closteroviridae, while P9 orthologs are found in all Criniviruses. In this study, the expression of LIYV P5 and P9 proteins was confirmed; P5 is further characterized as an endoplasmic reticulum (ER)-localized integral transmembrane protein and P9 is a soluble protein. The knockout LIYV mutants presented reduced symptom severity and virus accumulation in Nicotiana benthamiana or lettuce plants, indicating their importance in efficient virus infection. The P5 mutant was successfully complemented by a dislocated P5 in the LIYV genome. The structural regions of P5 were tested and all were found to be required for the appropriate functions of P5. In addition, P5, as well as its ortholog P6, encoded by Citrus tristeza virus (CTV) and another ER-localized protein encoded by LIYV RNA1, were found to cause cell death when expressed in N. benthamiana plants from a TMV vector, and induce ER stress and the unfolded protein response (UPR).

Accumulation of 24 nucleotide transgene-derived siRNAs is associated with crinivirus immunity in transgenic plants.

RNA silencing is a conserved antiviral defence mechanism that has been used to develop robust resistance against plant virus infections. Previous efforts have been made to develop RNA silencing-mediated resistance to criniviruses, yet none have given immunity. In this study, transgenic Nicotiana benthamiana plants harbouring a hairpin construct of the Lettuce infectious yellows virus (LIYV) RNA-dependent RNA polymerase (RdRp) sequence exhibited immunity to systemic LIYV infection. Deep sequencing analysis was performed to characterize virus-derived small interfering RNAs (vsiRNAs) generated on systemic LIYV infection in non-transgenic N. benthamiana plants as well as transgene-derived siRNAs (t-siRNAs) derived from the immune-transgenic plants before and after LIYV inoculation. Interestingly, a similar sequence distribution pattern was obtained with t-siRNAs and vsiRNAs mapped to the transgene region in both immune and susceptible plants, except for a significant increase in t-siRNAs of 24 nucleotides in length, which was consistent with small RNA northern blot results that showed the abundance of t-siRNAs of 21, 22 and 24 nucleotides in length. The accumulated 24-nucleotide sequences have not yet been reported in transgenic plants partially resistant to criniviruses, and thus may indicate their correlation with crinivirus immunity. To further test this hypothesis, we developed transgenic melon (Cucumis melo) plants immune to systemic infection of another crinivirus, Cucurbit yellow stunting disorder virus (CYSDV). As predicted, the accumulation of 24-nucleotide t-siRNAs was detected in transgenic melon plants by northern blot. Together with our findings and previous studies on crinivirus resistance, we propose that the accumulation of 24-nucleotide t-siRNAs is associated with crinivirus immunity in transgenic plants.

Efficient Protein Expression and Virus-Induced Gene Silencing in Plants Using a Crinivirus-Derived Vector.

Plant virus-based vectors are valuable tools for recombinant gene expression and functional genomics for both basic and applied research. In this study, Lettuce infectious yellows virus (LIYV) of the genus Crinivirus was engineered into a virus vector that is applicable for efficient protein expression and virus-induced gene silencing (VIGS) in plants. We examined gene replacement and “add a gene” strategies to develop LIYV-derived vectors for transient expression of the green fluorescent protein (GFP) reporter in Nicotiana benthamiana plants. The latter yielded higher GFP expression and was further examined by testing the effects of heterologous controller elements (CEs). A series of five vector constructs with progressively extended LIYV CP sgRNA CEs were tested, the longest CE gave the highest GFP expression but lower virus accumulation. The whitefly transmissibility of the optimized vector construct to other host plants, and the capability to accommodate and express a larger gene, a 1.8 kb β-glucuronidase (GUS) gene, were confirmed. Furthermore, the LIYV vector was also validated VIGS by silencing the endogenous gene, phytoene desaturase (PDS) in N. benthamiana plants, and the transgene GFP in N. benthamiana line 16c plants. Therefore, LIYV-derived vectors could provide a technical reference for developing vectors of other economically important criniviruses.

Inspirations on Virus Replication and Cell-to-Cell Movement from Studies Examining the Cytopathology Induced by Lettuce infectious yellows virus in Plant Cells.

Lettuce infectious yellows virus (LIYV) is the type member of the genus Crinivirus in the family Closteroviridae. Like many other positive-strand RNA viruses, LIYV infections induce a number of cytopathic changes in plant cells, of which the two most characteristic are: Beet yellows virus-type inclusion bodies composed of vesicles derived from cytoplasmic membranes; and conical plasmalemma deposits (PLDs) located at the plasmalemma over plasmodesmata pit fields. The former are not only found in various closterovirus infections, but similar structures are known as ‘viral factories’ or viroplasms in cells infected with diverse types of animal and plant viruses. These are generally sites of virus replication, virion assembly and in some cases are involved in cell-to-cell transport. By contrast, PLDs induced by the LIYV-encoded P26 non-virion protein are not involved in replication but are speculated to have roles in virus intercellular movement. These deposits often harbor LIYV virions arranged to be perpendicular to the plasma membrane over plasmodesmata, and our recent studies show that P26 is required for LIYV systemic plant infection. The functional mechanism of how LIYV P26 facilitates intercellular movement remains unclear, however, research on other plant viruses provides some insights on the possible ways of viral intercellular movement through targeting and modifying plasmodesmata via interactions between plant cellular components and viral-encoded factors. In summary, beginning with LIYV, we review the studies that have uncovered the biological determinants giving rise to these cytopathological effects and their importance in viral replication, virion assembly and intercellular movement during the plant infection by closteroviruses, and compare these findings with those for other positive-strand RNA viruses.

Whitefly feeding behavior and retention of a foregut-borne crinivirus exposed to artificial diets with different pH values.

Transmission of plant viruses by phytophagous hemipteran insects encompasses complex interactions underlying a continuum of processes involved in virus acquisition, retention and inoculation combined with vector feeding behavior. Here, we investigated the effects of dietary pH on whitefly (Bemisia tabaci) feeding behavior and release of Lettuce infectious yellows virus (LIYV) virions retained in the vector's foregut. Electrical penetration graph analysis revealed that variables associated with whitefly probing and ingestion did not differ significantly in pH (4, 7.4, and 9) adjusted artificial diets. To investigate virus retention and release, whiteflies allowed to acquire LIYV virions in a pH 7.4 artificial diet were fed pH 4, 7.4, or 9 virion-free artificial (clearing) diets. Immunofluorescent localization analyses indicated that virions remained bound to the foreguts of approximately 20%-24% of vectors after they fed on each of the 3 pH-adjusted clearing diets. When RNA preparations from the clearing diets were analyzed by reverse transcription (RT) nested-PCR and, in some cases, real-time qPCR, successful amplification of LIYV-specific sequence was infrequent but consistently repeatable for the pH 7.4 diet but never observed for the pH 4 and 9 diets, suggesting a weak pH-dependent effect for virion release. Viruliferous vectors that fed on each of the 3 pH-adjusted clearing diets transmitted LIYV to virus-free plants. These results suggest that changes in pH values alone in artificial diet do not result in observable changes in whitefly feeding behaviors, an observation that marks a first in the feeding of artificial diet by whitefly vectors; and that there is a potential causal and contingent relationship between the pH in artificial diet and the release/inoculation of foregut bound virions.

Whitefly-transmitted criniviruses of cucurbits: current status and future prospects.

In the past decade, crinviruses have gained interest due to their rapid widespread and destructive nature for cucurbit cultivation. Several members of the genus Crinivirus are considered emerging viruses. Currently, four criniviruses: Beet pseudo-yellows virus, Cucurbit chlorotic yellows virus, Cucurbit yellow stunting disorder virus and Lettuce infectious yellows virus have been reported to infect field- or greenhouse- grown cucurbits. Apart from their cucurbit hosts, criniviruses infect other cash crops and weeds. Criniviruses are exclusively transmitted by whiteflies. The virion titer and the vector genus or species complex are predominant factors affecting virus transmission. These criniviruses maintain genetic stability with limited intra-species variability. They share similar core genome structure and replication strategies with some variations in the non-core proteins and downstream replication processes. Management of the diseases induced by criniviruses relies on integrated disease management strategies and on resistant varieties, when available. This review will cover their epidemiology, molecular biology, detection and management.

Crinivirus replication and host interactions.

Criniviruses comprise one of the genera within the family Closteroviridae. Members in this family are restricted to the phloem and rely on whitefly vectors of the genera Bemisia and/or Trialeurodes for plant-to-plant transmission. All criniviruses have bipartite, positive-sense single-stranded RNA genomes, although there is an unconfirmed report of one having a tripartite genome. Lettuce infectious yellows virus (LIYV) is the type species of the genus, the best studied so far of the criniviruses and the first for which a reverse genetics system was developed. LIYV RNA 1 encodes for proteins predicted to be involved in replication, and alone is competent for replication in protoplasts. Replication results in accumulation of cytoplasmic vesiculated membranous structures which are characteristic of most studied members of the Closteroviridae. These membranous structures, often referred to as Beet yellows virus (BYV)-type vesicles, are likely sites of RNA replication. LIYV RNA 2 is replicated in trans when co-infecting cells with RNA 1, but is temporally delayed relative to RNA 1. Efficient RNA 2 replication also is dependent on the RNA 1-encoded RNA-binding protein, P34. No LIYV RNA 2-encoded proteins have been shown to affect RNA replication, but at least four, CP (major coat protein), CPm (minor coat protein), Hsp70h, and P59 are virion structural components and CPm is a determinant of whitefly transmissibility. Roles of other LIYV RNA 2-encoded proteins are largely as yet unknown, but P26 is a non-virion protein that accumulates in cells as characteristic plasmalemma deposits which in plants are localized within phloem parenchyma and companion cells over plasmodesmata connections to sieve elements. The two remaining crinivirus-conserved RNA 2-encoded proteins are P5 and P9. P5 is 39 amino acid protein and is encoded at the 5′ end of RNA 2 as ORF 1 and is part of the hallmark closterovirus gene array. The orthologous gene in BYV has been shown to play a role in cell-to-cell movement and indicated to be localized to the endoplasmic reticulum as a Type III integral membrane protein. The other small protein, P9, is encoded by ORF 4 overlaps with ORF 3 that encodes the structural protein, P59. P9 seems to be unique to viruses in the genus Crinivirus, as no similar protein has been detected in viruses of the other two genera of the Closteroviridae.

A Quantum Dot-Immunofluorescent Labeling Method to Investigate the Interactions between a Crinivirus and Its Whitefly Vector.

Successful vector-mediated plant virus transmission entails an intricate but poorly understood interplay of interactions among virus, vector, and plant. The complexity of interactions requires continually improving/evaluating tools and methods for investigating the determinants that are central to mediating virus transmission. A recent study using an organic fluorophore (Alexa Fluor)-based immunofluorescent localization assay demonstrated that specific retention of Lettuce infectious yellows virus (LIYV) virions in the anterior foregut or cibarium of its whitefly vector is required for virus transmission. Continuous exposure of organic fluorophore to high excitation light intensity can result in diminished or loss of signals, potentially confounding the identification of important interactions associated with virus transmission. This limitation can be circumvented by incorporation of photostable fluorescent nanocrystals, such as quantum dots (QDs), into the assay. We have developed and evaluated a QD-immunofluorescent labeling method for the in vitro and in situ localization of LIYV virions based on the recognition specificity of streptavidin-conjugated QD605 (S-QD605) for biotin-conjugated anti-LIYV IgG (B-αIgG). IgG biotinylation was verified in a blot overlay assay by probing SDS-PAGE separated B-αIgG with S-QD605. Immunoblot analyses of LIYV using B-αIgG and S-QD605 resulted in a virus detection limit comparable to that of DAS-ELISA. In membrane feeding experiments, QD signals were observed in the anterior foregut or cibarium of virion-fed whitefly vectors but absent in those of virion-fed whitefly non-vectors. Specific virion retention in whitefly vectors corresponded with successful virus transmission. A fluorescence photobleaching assay of viruliferous whiteflies fed B-αIgG and S-QD605 vs. those fed anti-LIYV IgG and Alexa Fluor 488-conjugated IgG revealed that QD signal was stable and deteriorated approx. seven- to eight-fold slower than that of Alexa Fluor.

Acquisition of Lettuce infectious yellows virus by Bemisia tabaci perturbs the transmission of Lettuce chlorosis virus

Viruses in the genus Crinivirus infect diverse plant species and are transmitted by specific whitefly vectors, but the basis for vector specific transmission remains poorly understood. Here, we demonstrated that purified virion preparations of Lettuce chlorosis virus (LCV) contained filamentous particles that were consistently transmitted to plants by whiteflies (Bemisia tabaci biotypes A and B) following membrane feeding, suggesting that the preparations contained biologically active virions with all the components essential for specific vector transmission. We also demonstrated in sequential membrane feeding experiments that B. tabaci biotype A pre-fed with high concentrations of Lettuce infectious yellows virus (LIYV) virions followed by decreasing concentrations of LCV virions either abolished or interfered with the transmission of the latter. However, in the reverse treatment, an abolishment/interference in transmission of LIYV was not observed. These results suggest that both viruses share a common transmission pathway in B. tabaci biotype A, and factors other than virion quality and quantity may additionally influence their transmission. To begin investigating the viral determinants that are involved in mediating the whitefly transmission of LCV, virions were analyzed by Western immunoblotting. Our results showed that virions were positively identified by antisera produced against three E. coli expressed recombinant LCV capsid proteins—the major coat protein [CP], minor CP [CPm], and P60.

Lettuce infectious yellows virus (LIYV) RNA 1-encoded P34 is an RNA-binding protein and exhibits perinuclear localization

The Crinivirus, Lettuce infectious yellows virus (LIYV) has a bipartite, positive-sense ssRNA genome. LIYV RNA 1 encodes replication-associated proteins while RNA 2 encodes proteins needed for other aspects of the LIYV life cycle. LIYV RNA 1 ORF 2 encodes P34, a trans enhancer for RNA 2 accumulation. Here we show that P34 is a sequence non-specific ssRNA-binding protein in vitro. P34 binds ssRNA in a cooperative manner, and the C-terminal region contains the RNA-binding domain. Topology predictions suggest that P34 is a membrane-associated protein and the C-terminal region is exposed outside of the membrane. Furthermore, fusions of P34 to GFP localized to the perinuclear region of transfected protoplasts, and colocalized with an ER-specific dye. This localization was of interest since LIYV RNA 1 replication (with or without P34 protein) induced strong ER rearrangement to the perinuclear region. Together, these data provide insight into LIYV replication and possible functions of P34.

Two Crinivirus-specific proteins of Lettuce infectious yellows virus (LIYV), P26 and P9, are self-interacting

Interactions of Lettuce infectious yellows virus (LIYV)-encoded proteins were tested by yeast-two-hybrid (Y2H) assays. LIYV-encoded P34, Hsp70h, P59, CP, CPm, and P26 were tested in all possible pairwise combinations. Interaction was detected only for the P26–P26 combination. P26 self-interaction domains were mapped using a series of N- and C-terminal truncations. Orthologous P26 proteins from the criniviruses Beet pseudoyellows virus (BPYV), Cucurbit yellow stunting disorder virus (CYSDV), and Lettuce chlorosis virus (LCV) were also tested, and each exhibited strong self-interaction but no interaction with orthologous proteins. Two small putative proteins encoded by LIYV RNA2, P5 and P9, were also tested for interactions with the six aforementioned LIYV proteins and each other. No interactions were detected for P5, but P9–P9 self-interaction was detected. P26- and P9-encoding genes are present in all described members of the genus Crinivirus, but are not present in other members of the family Closteroviridae. LIYV P26 has previously been demonstrated to induce a unique LIYV cytopathology, plasmalemma deposits (PLDs), but no role is yet known for P9.

Agroinoculation of the Crinivirus, Lettuce infectious yellows virus, for systemic plant infection.

Lettuce infectious yellows virus (LIYV) is phloem-limited, non-mechanically transmissible, and is transmitted to plants only by Bemisia tabaci. Here, we developed agroinoculation to deliver LIYV to plants thereby obviating the need for B. tabaci. Agroinfiltration of RNA 1 containing a green fluorescent protein gene into Nicotiana benthamiana leaves resulted in subliminal infections, as judged by green fluorescence. Agroinfiltration of LIYV wild-type RNA 1 and 2 constructs resulted in systemic infections in N. benthamiana plants and typical LIYV symptoms. In addition, partially purified LIYV virions from agroinoculated N. benthamiana plants were successfully acquired via membrane-feeding and transmitted to lettuce plants by B. tabaci. Agroinoculation coupled with targeted mutagenesis technologies will greatly enhance LIYV reverse genetics studies to characterize LIYV gene functions in planta for processes such as virus replication, recombination, trafficking, symptom elicitation and virus-vector interactions.

Further complexity of the genus Crinivirus revealed by the complete genome sequence of Lettuce chlorosis virus (LCV) and the similar temporal accumulation of LCV genomic RNAs 1 and 2.

The sequence of Lettuce chlorosis virus (LCV) (genus Crinivirus) was determined and found to contain unique open reading frames (ORFs) and ORFs similar to those of other criniviruses, as well as 3' non-coding regions that shared a high degree of identity. Northern blot analysis of RNA extracted from LCV-infected plants identified subgenomic RNAs corresponding to six prominent internal ORFs and detected several novel LCV-single stranded RNA species. Virus replication in tobacco protoplasts was investigated and results indicated that LCV replication proceeded with novel crinivirus RNA accumulation kinetics, wherein viral genomic RNAs exhibited a temporally similar expression pattern early in the infection. This was noticeably distinct from the asynchronous RNA accumulation pattern previously observed for Lettuce infectious yellows virus (LIYV), the type member of the genus, suggesting that replication of the two viruses likely operate via dissimilar mechanisms.

Synergistic interaction between the Potyvirus, Turnip mosaic virus and the Crinivirus, Lettuce infectious yellows virus in plants and protoplasts

Lettuce infectious yellows virus (LIYV), the type member of the genus Crinivirus in the family Closteroviridae, is specifically transmitted by the sweet potato whitefly ( Bemisia tabaci) in a semipersistent manner. LIYV infections result in a low virus titer in plants and protoplasts, impeding reverse genetic efforts to analyze LIYV gene/protein functions. We found that synergistic interactions occurred in mixed infections of LIYV and Turnip mosaic virus (TuMV) in Nicotiana benthamiana plants, and these resulted in enhanced accumulation of LIYV. Furthermore, we examined the ability of transgenic plants and protoplasts expressing only the TuMV P1/HC-Pro sequence to enhance the accumulation of LIYV. LIYV RNA and protein titers increased by as much as 8-fold in these plants and protoplasts relative to control plants. LIYV infections remained phloem-limited in P1/HC-Pro transgenic plants, suggesting that enhanced accumulation of LIYV in these plants was due primarily to increased replication efficiency, not to greater spread.

Lettuce infectious yellows virus-encoded P26 induces plasmalemma deposit cytopathology

Lettuce infectious yellows virus (LIYV) encodes a 26 kDa protein (P26) previously shown to associate with plasmalemma deposits (PLDs), unique LIYV-induced cytopathologies located at the plasmalemma over plasmodesmata pit fields in companion cells and phloem parenchyma. To further characterize the relationship of P26 and PLDs, we assessed localization and cytopathology induction of P26 expressed from either LIYV or a heterologous Tobacco mosaic virus (TMV) vector using green fluorescent protein (GFP) fusions, immunofluorescence microscopy, biochemical fractionation, and transmission electron microscopy (TEM). TEM analyses demonstrated that P26 not only associated with, but induced formation of PLDs in the absence of other LIYV proteins. Interestingly, PLDs induced by P26-expressing TMV were no longer confined to phloem cells. Putative P26 orthologs from two other members of the genus Crinivirus which do not induce conspicuous PLDs exhibited fractionation properties similar to LIYV P26 but were not associated with any PLD-like cytopathology.