Identification of domains of the Tomato spotted wilt virus NSm protein involved in tubule formation, movement and symptomatology

AbstractDeletion and alanine-substitution mutants of the Tomato spotted wilt virus NSm protein were generated to identify domains involved in tubule formation, movement and symptomatology using a heterologous Tobacco mosaic virus expression system. Two regions of NSm, G19-S159 and G209-V283, were required for both tubule formation in protoplasts and cell-to-cell movement in plants, indicating a correlation between these activities. Three amino acid groups, D154, EYKK205–208 and EEEEE284–288 were linked with long-distance movement in Nicotiana benthamiana. EEEEE284–288 was essential for NSm-mediated long-distance movement, whereas D154 was essential for tubule formation and cell-to-cell movement; indicating separate genetic controls for cell-to-cell and long-distance movement. The region I57-N100 was identified as the determinant of foliar necrosis in Nicotiana benthamiana, and mutagenesis of HH93–94 greatly reduced necrosis. These findings are likely applicable to other tospovirus species, especially those within the ‘New World’ group as NSm sequences are highly conserved

Accumulation of a 5′ proximal subgenomic RNA of \u3ci\u3eCitrus tristeza virus\u3c/i\u3e is correlated with encapsidation by the minor coat protein

During replication, Citrus tristeza virus (CTV) produces large amounts of two unusual subgenomic (sg) RNAs that are positive-stranded and 5′ coterminal. Although these RNAs are produced in similar amounts and are similar in size, with LMT1 (~750 nt) only slightly larger than LMT2 (~650), we found that the similar sgRNAs are produced differently. We previously showed that the LMT1 RNA is produced by premature termination during genomic RNA synthesis. However, LMT2 production was found to correlate with virion assembly instead of RNA replication. The time course of accumulation of the LMT2 RNA occurred late, coinciding with virion accumulation. The long flexuous virions of CTV contain two coat proteins that encapsidate the virions in a polar manner. The major coat protein encapsidates ~97% of the virion, while the minor capsid protein encapsidates the remainder of the genome beginning in the 5′ non-translated region with the transition zone at ~630 nucleotides from the 5′ end. The section of the virion RNA that was encapsidated by CPm was identical in size to the LMT2 RNA, suggesting that the LMT2 RNA represented a portion of the viral RNA protected by CPm encapsidation. Mutations that abrogated encapsidation by CPm also abolished the accumulation of LMT2 RNA. Thus, these two unusual but similar RNAs are produced via different pathways, one from RNA replication and one processed by the virion assembly process. To our knowledge, this represents the first evidence of a viral RNA processed by the assembly mechanism

Molecular Characterization of \u3ci\u3eCitrus tatter leaf virus\u3c/i\u3e Historically Associated with Meyer Lemon Trees: Complete Genome Sequence and Development of Biologically Active In Vitro Transcripts

Citrus tatter leaf virus isolated from Meyer lemon trees (CTLV-ML) from California and Florida induces bud union incompatibility of citrus trees grafted on the widely used trifoliate and trifoliate hybrid rootstocks. The complete genome sequence of CTLV-ML was determined to be 6,495 nucleotides (nts), with two overlapping open reading frames (ORFs) and a poly (A) tail at the 3′ end. The genome organization is similar to other capilloviruses, with ORF1 (nts 37 to 6,354) encoding a putative 242-kDa polyprotein which contains replication-associated domains plus a coat protein (CP), and ORF2 (nts 4,788 to 5,750), which is located within ORF1 in a different reading frame and encodes a putative movement protein. Although the proteins encoded by CTLV-ML possesses 84 to 96% amino acid sequence identity with strains of Apple stem grooving virus (ASGV), we observed two strikingly different regions in ORF1: variable region I (amino acids 532 to 570) and variable region II (amino acids 1,583 to 1,868), with only 15 to 18 and 56 to 62% identities, respectively, with the corresponding regions of ASGV strains. Conditions for a herbaceous systemic assay host were optimized in which the wildtype virus induced systemic infection in Phaseolus vulgaris cv. Light Red Kidney (LRK) bean plants at 19 or 22°C but not at higher temperatures. In vitro transcripts generated from full-length cDNA clones induced systemic symptoms on LRK bean plants similar to that of the wild-type virus. Replication of the recombinant virus was confirmed by hybridization of a 5′ positive-stranded RNA-specific probe to a genome-sized RNA and by reverse-transcription polymerase chain reaction

Characterization of the 5′- and 3′-Terminal Subgenomic RNAs Produced by a Capillovirus: Evidence for a CP Subgenomic RNA

The members of Capillovirus genus encode two overlapping open reading frames (ORFs): ORF1 encodes a large polyprotein containing the replication-associated proteins plus a coat protein (CP), and ORF2 encodes a movement protein (MP), located within ORF1 in a different reading frame. Organization of the CP sequence as part of the replicase ORF is unusual in capilloviruses. In this study, we examined the capillovirus genome expression strategy by characterizing viral RNAs produced by Citrus tatter leaf virus (CTLV), isolate ML, a Capillovirus. CTLV-ML produced a genome-length RNA of ∼6.5-kb and two 3′-terminal sgRNAs in infected tissue that contain the MP and CP coding sequences (3′-sgRNA1), and the CP coding sequence (3′-sgRNA2), respectively. Both 3′-sgRNAs initiate at a conserved octanucleotide (UUGAAAGA), and are 1826 (3′-sgRNA1) and 869 (3′-sgRNA2) nts with 119 and 15 nt leader sequences, respectively, suggesting that these two 3′- sgRNAs could serve to express the MP and CP. Additionally, accumulation of two 5′-terminal sgRNAs of 5586 (5′-sgRNA1) and 4625 (5′-sgRNA2) nts was observed, and their 3′-termini mapped to 38–44 nts upstream of the transcription start sites of 3′-sgRNAs. The presence of a separate 3′-sgRNA corresponding to the CP coding sequence and its cognate 5′-terminal sgRNA (5′-sgRNA1) suggests that CTLV-ML produces a dedicated sg mRNA for the expression of its CP

Persistent Infection and Promiscuous Recombination of Multiple Genotypes of an RNA Virus within a Single Host Generate Extensive Diversity

Recombination and reassortment of viral genomes are major processes contributing to the creation of new, emerging viruses. These processes are especially significant in long-term persistent infections where multiple viral genotypes co-replicate in a single host, generating abundant genotypic variants, some of which may possess novel host-colonizing and pathogenicity traits. In some plants, successive vegetative propagation of infected tissues and introduction of new genotypes of a virus by vector transmission allows for viral populations to increase in complexity for hundreds of years allowing co-replication and subsequent recombination of the multiple viral genotypes. Using a resequencing microarray, we examined a persistent infection by a Citrus tristeza virus (CTV) complex in citrus, a vegetatively propagated, globally important fruit crop, and found that the complex comprised three major and a number of minor genotypes. Subsequent deep sequencing analysis of the viral population confirmed the presence of the three major CTV genotypes and, in addition, revealed that the minor genotypes consisted of an extraordinarily large number of genetic variants generated by promiscuous recombination between the major genotypes. Further analysis provided evidence that some of the recombinants underwent subsequent divergence, further increasing the genotypic complexity. These data demonstrate that persistent infection of multiple viral genotypes within a host organism is sufficient to drive the large-scale production of viral genetic variants that may evolve into new and emerging viruses

Colonization of Seeds of Citrus Rootstock Varieties by ‘Candidatus Liberibacter asiaticus’

Huanglongbing (HLB) is a disease of citrus associated with a systemic infection by the α-proteobacterium ‘Candidatus Liberibacter asiaticus’.  Infection of an individual tree can occur via psyllids (Diaphorina citri Kuwayama) carrying the bacterium or if the tree is propagated from infected budwood.  Seed transmission is another possible mode of dissemination of the pathogen.  Rootstock varieties are propagated from seed so we assessed the seed transmission among eighteen rootstock varieties using seeds from mature fruit collected in late winter and immature fruit collected in late summer.  In dissected seeds real-time PCR detected pathogen DNA in seed coats at an incidence of 0-100%, whereas no pathogen DNA was detected in cotyledons or embryos from any variety.  Seeds collected in late winter were germinated in a greenhouse and no pathogen DNA was detected in extracts of shoots of 425 seedlings harvested at 7-10 days post-germination whereas a small amount of pathogen DNA was detected in extracts from 6 of 425 roots. All six positive samples were the same rootstock variety, which had 100% colonization of seed coats.  The positive samples likely are a result of remnant seed coat, which was not completely removed from cotyledons of the harvested seedlings.  The data from this study suggest the pathogen can colonize the seed coat but it does not colonize embryos, which makes seed transmission unlikely.&nbsp

Colonization of Seeds of Citrus Rootstock Varieties by ‘Candidatus Liberibacter asiaticus’

Huanglongbing (HLB) is a disease of citrus associated with a systemic infection by the α-proteobacterium ‘Candidatus Liberibacter asiaticus’.  Infection of an individual tree can occur via psyllids (Diaphorina citri Kuwayama) carrying the bacterium or if the tree is propagated from infected budwood.  Seed transmission is another possible mode of dissemination of the pathogen.  Rootstock varieties are propagated from seed so we assessed the seed transmission among eighteen rootstock varieties using seeds from mature fruit collected in late winter and immature fruit collected in late summer.  In dissected seeds real-time PCR detected pathogen DNA in seed coats at an incidence of 0-100%, whereas no pathogen DNA was detected in cotyledons or embryos from any variety.  Seeds collected in late winter were germinated in a greenhouse and no pathogen DNA was detected in extracts of shoots of 425 seedlings harvested at 7-10 days post-germination whereas a small amount of pathogen DNA was detected in extracts from 6 of 425 roots. All six positive samples were the same rootstock variety, which had 100% colonization of seed coats.  The positive samples likely are a result of remnant seed coat, which was not completely removed from cotyledons of the harvested seedlings.  The data from this study suggest the pathogen can colonize the seed coat but it does not colonize embryos, which makes seed transmission unlikely.&nbsp

Validation of ‘Variable Number of Tandem Repeat’-Based Approach for Examination of ‘<i>Candidatus</i> Liberibacter asiaticus’ Diversity and Its Applications for the Analysis of the Pathogen Populations in the Areas of Recent Introduction

<div><p>Citrus greening (Huanglongbing, HLB) is one of the most destructive diseases of citrus worldwide. In South Asia HLB has been known for more than a century, while in Americas the disease was found relatively recently. HLB is associated with three species of ‘<i>Candidatus</i> Liberibacter’ among which ‘<i>Ca.</i> Liberibacter asiaticus’ (<i>C</i>Las) has most wide distribution. Recently, a number of studies identified different regions in the <i>C</i>Las genome with variable number of tandem repeats (VNTRs) that could be used for examination of <i>C</i>Las diversity. One of the objectives of the work presented here was to further validate the VNTR analysis-based approach by assessing the stability of these repeats upon multiplication of the pathogen in a host over an extended period of time and upon its passaging from a host to a host using <i>C</i>Las populations from Florida. Our results showed that the numbers of tandem repeats in the four <i>loci</i> tested display very distinguishable “signature profiles” for the two Florida-type <i>C</i>Las haplotype groups. Remarkably, the profiles do not change upon passage of the pathogen in citrus and psyllid hosts as well as after its presence within a host over a period of five years, suggesting that VNTR analysis-based approach represents a valid methodology for examination of the pathogen populations in various geographical regions. Interestingly, an extended analysis of <i>C</i>Las populations in different locations throughout Florida and in several countries in the Caribbean and Central America regions and in Mexico where the pathogen has been introduced recently demonstrated the dispersion of the same haplotypes of <i>C</i>Las. On the other hand, these CLas populations appeared to differ significantly from those obtained from locations where the disease has been present for a much longer time.</p></div