An engineered citrus tristeza virus (T36CA)-based vector induces gene-specific RNA silencing and is graft transmissible to commercial citrus varieties.

A protein-expressing citrus tristeza virus (CTV)-based vector construct, pT36CA-V1.3, obtained from a California isolate of the T36 strain (T36CA), was retooled into a virus induced gene silencing (VIGS) system intended for use with studies of California citrus. VIGS constructs engineered with a truncated Citrus macrophylla (Cm) PHYTOENE DESATURASE (PDS) gene sequence in the sense or anti-sense orientation worked equally well to silence the endogenous CmPDS gene. In a parallel effort to optimize vector performance, two non-synonymous nucleotides in open reading frame 1a of pT36CA-V1.3 were replaced with those conserved in the reference sequences from the T36CA cDNA library. The resulting viruses, T36CA-V1.4 (with one amino acid modification: D760N) and T36CA-V1.5 (with two amino acid modifications: D760N and P1174L), along with T36CA-V1.3 were individually propagated in Nicotiana benthamiana and C. macrophylla plants. Enzyme-linked immunosorbent assay (ELISA) measurements of extracts of the newly emerged leaves suggested that all three viruses accumulated to similar levels in N. benthamiana plants at 5 week-post-inoculation. ELISA values of T36CA-V1.4- and -V1.5-infected C. macrophylla samples were significantly higher than that of T36CA-V1.3-infected samples within an 8 to 12 month-post-inoculation (mpi) window, suggesting a higher accumulation of T36CA-V1.4 and -V1.5 than T36CA-V1.3. However, at 36 mpi, the ELISA values suggested that all three viruses accumulated to similar levels. When C. macrophylla plants infected with each of the three viruses were grafted to commercial citrus varieties, a limited number of receptor plants became infected, demonstrating a weak but nonetheless (the first) successful delivery of T36CA to California-grown commercial citrus.

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.

Comparative analysis identifies amino acids critical for citrus tristeza virus (T36CA) encoded proteins involved in suppression of RNA silencing and differential systemic infection in two plant species.

Complementary (c)DNA clones corresponding to the full-length genome of T36CA (a Californian isolate of Citrus tristeza virus with the T36 genotype), which shares 99.1% identity with that of T36FL (a T36 isolate from Florida), were made into a vector system to express the green fluorescent protein (GFP). Agroinfiltration of two prototype T36CA-based vectors (pT36CA) to Nicotiana benthamiana plants resulted in local but not systemic GFP expression/viral infection. This contrasted with agroinfiltration of the T36FL-based vector (pT36FL), which resulted in both local and systemic GFP expression/viral infection. A prototype T36CA systemically infected RNA silencing-defective N. benthamiana lines, demonstrating that a genetic basis for its defective systemic infection was RNA silencing. We evaluated the in planta bioactivity of chimeric pT36CA-pT36FL constructs and the results suggested that nucleotide variants in several open reading frames of the prototype T36CA could be responsible for its defective systemic infection. A single amino acid substitution in each of two silencing suppressors, p20 (S107G) and p25 (G36D), of prototype T36CA facilitated its systemic infectivity in N. benthamiana (albeit with reduced titre relative to that of T36FL) but not in Citrus macrophylla plants. Enhanced virus accumulation and, remarkably, robust systemic infection of T36CA in N. benthamiana and C. macrophylla plants, respectively, required two additional amino acid substitutions engineered in p65 (N118S and S158L), a putative closterovirus movement protein. The availability of pT36CA provides a unique opportunity for comparative analysis to identify viral coding and noncoding nucleotides or sequences involved in functions that are vital for in planta infection.

Nucleotide heterogeneity at the terminal ends of the genomes of two California Citrus tristeza virus strains and their complete genome sequence analysis.

BACKGROUND: The non-translated regions at the genome ends of RNA viruses serve diverse functions and can exhibit various levels of nucleotide (nt) heterogeneity. However, the extent of nt heterogeneity at the extreme termini of Citrus tristeza virus (CTV) genomes has not been comprehensively documented. This study aimed to characterize two widely prevalent CTV genotypes, T36-CA and T30-CA, from California that have not been sequenced or analyzed substantially. The information obtained will be used in our ongoing effort to construct the infectious complementary (c) DNA clones of these viruses. METHODS: The terminal nts of the viral genomes were identified by sequencing cDNA clones of the plus- and/or minus-strand of the viral double-stranded (ds) RNAs generated using 5' and 3' rapid amplification of cDNA ends. Cloned cDNAs corresponding to the complete genome sequences of both viruses were generated using reverse transcription-polymerase chain reactions, sequenced, and subjected to phylogenetic analysis. RESULTS: Among the predominant terminal nts identified, some were identical to the consensus sequences in GenBank, while others were different or unique. Remarkably, one of the predominant 5' nt variants of T36-CA contained the consensus nts "AATTTCAAA" in which a highly conserved cytidylate, seen in all other full-length T36 sequences, was absent. As expected, but never systematically verified before, unique variants with additional nt (s) incorporated upstream of the 5' terminal consensus nts of T36-CA and T30-CA were also identified. In contrast to the extreme 5' terminal nts, those at the extreme 3' termini of T36-CA and T30-CA were more conserved compared to the reference sequences, although nt variants were also found. Notably, an additional thymidylate at the extreme 3' end was identified in many T36-CA sequences. Finally, based on pairwise comparisons and phylogenetic analysis with multiple reference sequences, the complete sequences of both viruses were found to be highly conserved with those of the respective genotypes. CONCLUSIONS: The extreme terminal nts in the T36-CA and T30-CA genomes were identified, revealing new insights on the heterogeneity of these CTV genomic regions. T36-CA and T30-CA were the first and the second genotypes, respectively, of CTV originating from California to be completely sequenced and analyzed.

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.

Agroinoculation of the cloned infectious cDNAs of Lettuce chlorosis virus results in systemic plant infection and production of whitefly transmissible virions.

Lettuce chlorosis virus (LCV) is a single stranded, positive strand RNA virus that is solely transmitted by specific whitefly vectors (Bemisia tabaci biotypes A and B) but not by mechanical leaf-rub inoculation. The roles of viral encoded proteins involved in the infection cycle of LCV have not yet been characterized due to the lack of reverse genetic tools. We present here a report of the successful development of an Agrobacterium-mediated inoculation system for the cloned cDNA constructs of LCV. The cDNAs of both LCV RNAs 1 and 2 were engineered into binary vectors in which the expression of LCV RNAs was regulated under a Cauliflower mosaic virus (CaMV) 35S promoter. In addition, by engineering the sequence elements of the Hepatitis delta virus ribozyme and the nopaline synthase 3' untranslated region immediately downstream of the last nucleotide of LCV RNAs 1 and 2 in the binary vector constructs, the in planta produced LCV transcripts were expected to bear authentic 3' termini. Both constructs were transformed into Agrobacterium tumefaciens cells and infiltrated in Nicotiana benthamiana plants. Three to four weeks post-agroinoculation, the N. benthamiana plants developed typical interveinal chlorosis and LCV infection was detected in the systemic leaves by reverse transcription-PCR. Virions purified from the LCV-infected N. benthamiana plants were flexuous rod-shaped and were transmissible by both B. tabaci biotypes A and B following membrane feeding. These results support the conclusion that Agrobacterium-mediated inoculation of LCV binary vectors in N. benthamiana plants results in LCV infection and the production of biologically active, whitefly transmissible virions. This system represents an important tool for use with reverse genetics designed for the study of LCV gene functions.

Replication of Lettuce chlorosis virus (LCV), a crinivirus in the family Closteroviridae, is accompanied by the production of LCV RNA 1-derived novel RNAs.

Cloned infectious complementary DNAs of the bipartite genomic RNAs of Lettuce chlorosis virus (LCV) were constructed. Inoculation of tobacco protoplasts with the in vitro produced RNAs 1 and 2 transcripts, or with RNA 1 transcript alone, resulted in viral replication accompanied by the production of novel LCV RNA 1-derived RNAs. They included the abundantly accumulating LM-LCVR1-1 (~0.38 kb) and LM-LCVR1-2 (~0.3 kb), and the lowly accumulating HM-LCVR1-1 (~8.0 kb) and HM-LCVR1-2 (~6.6 kb), all of which reacted with riboprobes specific to the 5' end of RNA 1 in Northern blot analysis. LM-LCVR1-1 and HM-LCVR1-2 accumulated as positive-stranded RNAs that lacked complementary negative strands, while HM-LCVR1-1 and LM-LCVR1-2 accumulated in both polarities. Additional Northern blot, reverse transcription-polymerase chain reaction, cloning, and sequence analyses revealed LM-LCVR1-2 to be an authentic RNA 1-derived defective (D)RNA, suggesting that its synthesis and maintenance are supported in trans by an RNA 1 encoded replication machinery.

A virus capsid component mediates virion retention and transmission by its insect vector.

Numerous pathogens of humans, animals, and plants are transmitted by specific arthropod vectors. However, understanding the mechanisms governing these pathogen-vector interactions is hampered, in part, by the lack of easy-to-use analytical tools. We investigated whitefly transmission of Lettuce infectious yellows virus (LIYV) by using a unique immunofluorescent localization approach in which we fed virions or recombinant virus capsid components to whiteflies, followed by feeding them antibodies to the virions or capsid components, respectively. Fluorescent signals, indicating the retention of virions, were localized in the anterior foregut or cibarium of a whitefly vector biotype but not within those of a whitefly nonvector biotype. Retention of virions in these locations strongly corresponded with the whitefly vector transmission of LIYV. When four recombinant LIYV capsid components were individually fed to whitefly vectors, significantly more whiteflies retained the recombinant minor coat protein (CPm). As demonstrated previously and in the present study, whitefly vectors failed to transmit virions preincubated with anti-CPm antibodies but transmitted virions preincubated with antibodies recognizing the major coat protein (CP). Correspondingly, the number of insects that specifically retained virions preincubated with anti-CPm antibodies were significantly reduced compared with those that specifically retained virions preincubated with anti-CP antibodies. Notably, a transmission-defective CPm mutant was deficient in specific virion retention, whereas the CPm-restored virus showed WT levels of specific virion retention and transmission. These data provide strong evidence that transmission of LIYV is determined by a CPm-mediated virion retention mechanism in the anterior foregut or cibarium of whitefly vectors.

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.

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.