An Evolved 5' Untranslated Region of Alfalfa Mosaic Virus Allows the RNA Transport of Movement-Defective Variants.

Although the coat protein (CP) has a relevant role in the long-distance movement of alfalfa mosaic virus (AMV) and brome mosaic virus (BMV), its precise function is not fully understood. Previous results showed that a specific interaction between the C termini of the movement protein (MP) and the cognate CP is required for systemic transport. Thus, we have performed a compensatory evolution experiment using an AMV RNA3 derivative defective in long-distance transport that carries a BMV MP lacking the C-terminal 48 residues and unable to interact with the AMV CP. After several passages, five independent evolution lineages were able to move long distance. The analysis of the viral RNA of these lineages showed the presence of three different modifications located exclusively at the 5' untranslated region (5' UTR). The three evolved 5' UTR variants accumulated comparable levels of viral RNA and CP but reduced the accumulation of virus particles and the affinity between the 5' UTR and the AMV CP. In addition, the evolved 5' UTR increased cell-to-cell transport for both the AMV RNA3 carrying the BMV MP and that carrying the AMV MP. Finally, the evolved 5' UTRs allowed the systemic transport of an AMV RNA3 carrying a CP mutant defective in virus particles and increased the systemic transport of several AMV RNA3 derivatives carrying different viral MPs associated with the 30K superfamily. Altogether, our findings indicate that virus particles are not required for the systemic transport of AMV but also that BMV MP is competent for the short- and long-distance transport without the interaction with the CP. IMPORTANCE The results obtained in the present work could challenge the view of the role of the virus particle in the systemic transport of plant viruses. In this sense, we show that two different MPs are competent to systemically transport the AMV genome without the requirement of the virus particles, as reported for viruses lacking a CP (e.g., Umbravirus). The incapability of the viral MP to interact with the CP triggered virus variants that evolved to reduce the formation of virus particles, probably to increase the accessibility of the MP to the viral progeny. Our results point to the idea that virus particles would not be necessary for the viral systemic transport but would be necessary for vector virus transmission. This idea is reinforced by the observation that heterologous MPs also increased the systemic transport of the AMV constructs that have reduced encapsidation capabilities.

Negative modulation of SA signaling components by the capsid protein of tobacco mosaic virus is required for viral long-distance movement.

An important aspect of plant-virus interaction is the way viruses dynamically move over long distances and how plant immunity modulates viral systemic movement. Salicylic acid (SA), a well-characterized hormone responsible for immune responses against virus, is activated through different transcription factors including TGA and WRKY. In tobamoviruses, evidence suggests that capsid protein (CP) is required for long-distance movement, although its precise role has not been fully characterized yet. Previously, we showed that the CP of Tobacco Mosaic Virus (TMV)-Cg negatively modulates the SA-mediated defense. In this study, we analyzed the impact of SA-defense mechanism on the long-distance transport of a truncated version of TMV (TMV ∆CP virus) that cannot move to systemic tissues. The study showed that the negative modulation of NPR1 and TGA10 factors allows the long-distance transport of TMV ∆CP virus. Moreover, we observed that the stabilization of DELLA proteins promotes TMV ∆CP systemic movement. We also characterized a group of genes, part of a network modulated by CP, involved in TMV ∆CP long-distance transport. Altogether, our results indicate that CP-mediated downregulation of SA signaling pathway is required for the virus systemic movement, and this role of CP may be linked to its ability to stabilize DELLA proteins.

Comparative Anatomical Responses of Tolerant and Susceptible European Plum Varieties to Black Knot Disease.

Plums are affected by a cancerous disease called "black knot disease" caused by the fungus Apiosporina morbosa. It affects both Japanese (Prunus salicina) and European (Prunus domestica) plums equally. To understand the spread of the disease, histological analysis was performed in two different European plum cultivars (susceptible and tolerant). Light and scanning electron microscope (SEM) analyses confirmed the presence of the growing hyphae in the internal tissues of the susceptible trees. By using stereoscopic analysis with a fluorescence filter, we were able to detect the hyphae in the visible lesion area. At about 2 inches from above and below the knots, no spore or hypha were visible with the light microscope. However, SEM images showed strong evidence that the fungus is capable of migrating to adjacent vessels in the susceptible plum genotype. In fact, at that distance below and above the knots, conidia were detected inside xylem vessels suggesting a systemic movement of the fungus that has not been shown so far. No symptoms were observed in the resistant genotype. Starch granules, vessel occlusions, and lipid droplets were the main distinguishable characteristics between susceptible and tolerant varieties.

The 2b protein and C-terminal region of the 2a protein indispensably facilitate systemic movement of cucumber mosaic virus in radish with supplementary function by either the 3a or the coat protein.

BACKGROUND: In Raphanus sativus (Japanese radish), strain D8 of cucumber mosaic virus (CMV-D8) establishes a systemic infection and induces mild mosaic on upper, non-inoculated leaves, whereas strain Y of CMV (CMV-Y) causes only a local infection in the inoculated leaves. Here, we further analyzed the specific viral factor(s) of CMV-D8 that is (are) indispensable for systemic infection in Japanese radish. METHODS: To identify which genomic RNA(s) is (are) involved in systemic infection in radish, we carried out a pseudorecombination analysis between CMV-D8 and CMV-Y. With recombination analyses between CMV-D8 and CMV-Y using mutant/recombinant RNA2s, chimeric and point-mutated RNA3s, we identified viral factors that are indispensable for systemic infection. RESULTS: Viral RNA2 and RNA3 of CMV-D8 facilitated efficient virus spread into the upper, non-inoculated plant tissues of radish (cv. Tokinashi), but not those of CMV-Y. Recombinant RNA2s demonstrated that the 2b protein (2b) and the C-terminus of the 2a protein (2a) of CMV-D8 have a crucial role in systemic infection. In addition, we used chimeric and point-mutated RNA3s to that Pro17 and Pro129 in the coat protein (CP) of CMV-D8 are involved in efficient systemic infection and that Ser51 in the 3a protein (3a) of CMV-D8 has positive effects on systemic spread. The results suggested that these viral factors facilitate systemic infection of CMV-D8 in Japanese radish. CONCLUSION: The C-terminal region of 2a, the entire region of 2b, and supplementary function of either Ser51 in 3a or Pro17/Pro 129 in CP confer systemic infectivity on CMV-D8 in radish. These results further elucidate the complex interaction of viral proteins of CMV to complete systemic infection as a host-specific manner.

Raspberry leaf blotch emaravirus in Bosnia and Herzegovina: population structure and systemic movement.

Raspberry leaf blotch virus (RLBV) is the putative agent of the homonymous disease and even though Bosnia and Herzegovina is a major producer worldwide there is no report of the virus presence in the country. We studied the virus population structure and assessed its ability to move systemically. RLBV is widespread in production areas and has a homogeneous population structure; leading to the hypothesis that the primary mode of dissemination is propagation material. The ability of the virus to move systemically eliminates propagation of root cuttings as a viable option to obtain RLBV-free plants, leaving RT-PCR screening as the better option to propagate RLBV- free plants in the absence of clean-up facilities or certification programs in the country.

Amino acids at the exposed C-terminus of the S coat protein of cowpea mosaic virus play different roles in particle formation and viral systemic movement.

The icosahedral capsid of cowpea mosaic virus is formed by 60 copies of the large (L) and small (S) coat protein subunits. The 24-amino-acid C-terminal peptide of the S coat protein can undergo proteolytic cleavage without affecting particle stability or infectivity. Mutagenic studies have shown that this sequence is involved in particle assembly, virus movement, RNA encapsidation and suppression of gene silencing. However, it is unclear how these processes are related, and which part(s) of the sequence are involved in each process. Here, we have analysed the effect of mutations in the C-terminal region of the S protein on the assembly of empty virus-like particles and on the systemic movement of infectious virus. The results confirmed the importance of positively charged amino acids adjacent to the cleavage site for particle assembly and revealed that the C-terminal 11 amino acids are important for efficient systemic movement of the virus.

Persistence and transgenerational effect of plant-mediated RNAi in aphids.

Plant-mediated RNA interference (RNAi) has been successfully used as a tool to study gene function in aphids. The persistence and transgenerational effects of plant-mediated RNAi in the green peach aphid (GPA) Myzus persicae were investigated, with a focus on three genes with different functions in the aphid. Rack1 is a key component of various cellular processes inside aphids, while candidate effector genes MpC002 and MpPIntO2 (Mp2) modulate aphid-plant interactions. The gene sequences and functions did not affect RNAi-mediated down-regulation and persistence levels in the aphids. Maximal reduction of gene expression was ~70% and this was achieved at between 4 d and 8 d of exposure of the aphids to double-stranded RNA (dsRNA)-producing transgenic Arabidopsis thaliana. Moreover, gene expression levels returned to wild-type levels within ~6 d after removal of the aphids from the transgenic plants, indicating that a continuous supply of dsRNA is required to maintain the RNAi effect. Target genes were also down-regulated in nymphs born from mothers exposed to dsRNA-producing transgenic plants, and the RNAi effect lasted twice as long (12-14 d) in these nymphs. Investigations of the impact of RNAi over three generations of aphids revealed that aphids reared on dsMpC002 transgenic plants experienced a 60% decline in aphid reproduction levels compared with a 40% decline of aphids reared on dsRack1 and dsMpPIntO2 plants. In a field setting, a reduction of the aphid reproduction by 40-60% would dramatically decrease aphid population growth, contributing to a substantial reduction in agricultural losses.

Factors involved in the systemic transport of plant RNA viruses: the emerging role of the nucleus.

Compatible virus-host interactions depend on a suitable milieu in the host cells permitting viral gene expression, replication, and spread. During pathogenesis, viruses hijack the plant cellular machinery to access molecules, subcellular structures, and host transport pathways needed for infection. Vascular trafficking of virus transport forms (VTF) within the phloem is a crucial step in setting-up virus infection within the entire plant. Moreover, vascular trafficking is an essential step for the further transmission of the viruses by their natural vectors as movement of the viruses to the distant parts of the plant from the initial site of infection guarantees accessibility of the virus particle for vector transmission. With the recent advances in the field of plant virology several emerging themes of viral systemic movement occur linking the role of virus-mediated transcriptional reprogramming and nuclear factors in vascular trafficking. Recent studies have uncovered host factors involved in virus vascular trafficking. Surprisingly, it appears that the role of the nucleus and nuclear factors in virus movement is still under-appreciated. This review describes how these new themes started to emerge by using two contrasting modes of virus vascular trafficking. It is argued that the translocation of viral movement proteins into the nuclei is, in many cases, an essential step in promoting virus systemic infection.

The coat protein p25 from maize chlorotic mottle virus involved in symptom development and systemic movement of tobacco mosaic virus hybrids.

Viral coat protein (CP) has numerous critical functions in plant infection, but little is known about p25, the CP of maize chlorotic mottle virus (MCMV; Machlomovirus), which causes severe yield losses in maize worldwide. Here, we investigated the roles of p25 in pathogenicity and systemic movement, as well as potential interactions with host plants, using a hybrid tobacco mosaic virus (TMV)-based expression system. Highly conserved protein p25 is predicted to contain a membrane-anchored nuclear localization signal (NLS) sequence and an extracellular sequence. In transgenic Nicotiana benthamiana plants containing the movement protein (MP) of TMV (TMV-MP), p25 induced severe symptoms, including dwarf and foliar necrosis, and was detected in inoculated and non-inoculated leaves. After the deletion of NLS from nuclear-located p25, the protein was found throughout the host cell, and plant stunting and starch granule deformity were reduced. Systemic movement and pathogenicity were significantly impaired when the C-terminal regions of p25 were absent. Using virus-induced gene silencing (VIGS), the transcript level of heat shock protein HSP90 was distinctly lower in host plants in association with the absence of leaf necrosis induced by TMV-p25. Our results revealed crucial roles for MCMV p25 in viral pathogenicity, long-distance movement, and interactions with N. benthamiana.

Functional Analysis of V2 Protein of Beet Curly Top Iran Virus.

Geminivirus beet curly top Iran virus (BCTIV) is one of the main causal agents of the beet curly top disease in Iran and the newly established Becurtovirus genus type species. Although the biological features of known becurtoviruses are similar to those of curtoviruses, they only share a limited sequence identity, and no information is available on the function of their viral genes. In this work, we demonstrate that BCTIV V2, as the curtoviral V2, is also a local silencing suppressor in Nicotiana benthamiana and can delay the systemic silencing spreading, although it cannot block the cell-to-cell movement of the silencing signal to adjacent cells. BCTIV V2 shows the same subcellular localization as curtoviral V2, being detected in the nucleus and perinuclear region, and its ectopic expression from a PVX-derived vector also causes the induction of necrotic lesions in N. benthamiana, such as the ones produced during the HR, both at the local and systemic levels. The results from the infection of N. benthamiana with a V2 BCTIV mutant showed that V2 is required for systemic infection, but not for viral replication, in a local infection. Considering all these results, we can conclude that BCTIV V2 is a functional homologue of curtoviral V2 and plays a crucial role in viral pathogenicity and systemic movement.

Microtubules promote the non-cell autonomous action of microRNAs by inhibiting their cytoplasmic loading onto ARGONAUTE1 in Arabidopsis.

Mobile microRNAs (miRNAs) serve as local and long-distance signals in the developmental patterning and stress responses in plants. However, mechanisms governing the non-cell autonomous activities of miRNAs remain elusive. Here, we show that mutations that disrupt microtubule dynamics are specifically defective for the non-cell autonomous actions of mobile miRNAs, including miR165/6 that is produced in the endodermis and moves to the vasculature to pattern xylem cell fates in Arabidopsis roots. We show that KTN1, a subunit of a microtubule-severing enzyme, is required in source cells to inhibit the loading of miR165/6 into ARGONUATE1 (AGO1), which is cell autonomous, to enable the miRNA to exit the cell. Microtubule disruption enhances the association of miR165/6 with AGO1 in the cytoplasm. These findings suggest that although cell-autonomous miRNAs load onto AGO1 in the nucleus, the cytoplasmic AGO1 loading of mobile miRNAs is a key step regulated by microtubules to promote the range of miRNA cell-to-cell movement.

Genetic Dissection of the Erwinia amylovora Disease Cycle.

Fire blight, caused by the bacterial phytopathogen Erwinia amylovora, is an economically important and mechanistically complex disease that affects apple and pear production in most geographic production hubs worldwide. We compile, assess, and present a genetic outlook on the progression of an E. amylovora infection in the host. We discuss the key aspects of type III secretion-mediated infection and systemic movement, biofilm formation in xylem, and pathogen dispersal via ooze droplets, a concentrated suspension of bacteria and exopolysaccharide components. We present an overall outlook on the genetic elements contributing to E. amylovora pathogenesis, including an exploration of the impact of floral microbiomes on E. amylovora colonization, and summarize the current knowledge of host responses to an incursion and how this response stimulates further infection and systemic spread. We hope to facilitate the identification of new, unexplored areas of research in this pathosystem that can help identify evolutionarily susceptible genetic targets to ultimately aid in the design of sustainable strategies for fire blight disease mitigation.

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.

Effects of deletion at the TTTSTTT motif of Hibiscus latent Singapore virus coat protein on viral replication and long-distance movement.

Hibiscus latent Singapore virus (HLSV) mutant HLSV-22A could not express coat protein (CP) nor infect plants systemically (Niu et al., 2015). In this study, a serine- and threonine-rich motif TTTSTTT at the C-terminus of HLSV CP was found to be involved in virus replication and systemic movement. Deletion the last amino acid residue in HLSV-22A led to a more rapid virus replication, but with delayed systemic movement. When the RNA structure in TTTSTTT motif was altered, while keeping its amino acids unchanged, mutants HLSV-87A-mmSL and HLSV-22A-mmSL showed no change in viral replication. These results indicated that the unique TTTSTTT motif is associated with virus replication and systemic movement. Deletion but not substitution of amino acid(s) at the C-terminus of TTTSTTT motif of HLSV CP with short internal poly(A) track enhanced virus replication, whereas the virus with a longer internal poly(A) tract of 87 A showed delayed systemic movement (147 words).

The functional analysis of distinct tospovirus movement proteins (NSM) reveals different capabilities in tubule formation, cell-to-cell and systemic virus movement among the tospovirus species.

The lack of infectious tospovirus clones to address reverse genetic experiments has compromised the functional analysis of viral proteins. In the present study we have performed a functional analysis of the movement proteins (NSM) of four tospovirus species Bean necrotic mosaic virus (BeNMV), Chrysanthemum stem necrosis virus (CSNV), Tomato chlorotic spot virus (TCSV) and Tomato spotted wilt virus (TSWV), which differ biologically and molecularly, by using the Alfalfa mosaic virus (AMV) model system. All NSM proteins were competent to: i) support the cell-to-cell and systemic transport of AMV, ii) generate tubular structures on infected protoplast and iii) transport only virus particles. However, the NSM of BeNMV (one of the most phylogenetically distant species) was very inefficient to support the systemic transport. Deletion assays revealed that the C-terminal region of the BeNMV NSM, but not that of the CSNV, TCSV and TSWV NSM proteins, was dispensable for cell-to-cell transport, and that all the non-functional C-terminal NSM mutants were unable to generate tubular structures. Bimolecular fluorescence complementation analysis revealed that the C-terminus of the BeNMV NSM was not required for the interaction with the cognate nucleocapsid protein, showing a different protein organization when compared with other movement proteins of the '30K family'. Overall, our results revealed clearly differences in functional aspects among movement proteins from divergent tospovirus species that have a distinct biological behavior.

Resistance to Cucurbit yellow stunting disorder virus in Cucumber.

Three hundred accessions of Cucumis sativus, including wild cucumbers, land races, traditional cultivars, and breeding lines, were evaluated under natural-infection conditions in order to identify potential sources of resistance to Cucurbit yellow stunting disorder virus (CYSDV). Although 100% of the susceptible control plants showed typical yellowing symptoms induced by CYSDV, another 24 C. sativus accessions showed partial or total absence of yellowing symptoms. In contrast, when CYSDV inoculation was carried out under controlled conditions, only two (A1 and A2) of these 24 accessions showed resistance to the virus. The nature of the resistance found in A1 and A2 plants was characterized by studying the pattern of virus accumulation and symptom development under controlled infection conditions, and by analyzing the possible nonpreference of Bemisia tabaci for these accessions under free-choice conditions. There was a delay in the establishment of the CYSDV infection in A1 and A2 plants which was evident shortly after inoculation and in apical leaves of the plants at long times after inoculation. Symptom severity was also less for A1 and A2 than for a susceptible control at 8 and 12 weeks postinoculation. Thus, delayed viral infection appeared to be associated with decreased symptom severity in A1 and A2 plants. Our results also showed nonpreference for plants of the A2 accession by B. tabaci, the CYSDV vector.

A Viral Noncoding RNA Complements a Weakened Viral RNA Silencing Suppressor and Promotes Efficient Systemic Host Infection.

Systemic movement of beet necrotic yellow vein virus (BNYVV) in Beta macrocarpa depends on viral RNA3, whereas in Nicotiana benthamiana this RNA is dispensable. RNA3 contains a coremin motif of 20 nucleotides essential for the stabilization of noncoding RNA3 (ncRNA3) and for long-distance movement in Beta species. Coremin mutants that are unable to accumulate ncRNA3 also do not achieve systemic movement in Beta species. A mutant virus carrying a mutation in the p14 viral suppressor of RNA silencing (VSR), unable to move long distances, can be complemented with the ncRNA3 in the lesion phenotype, viral RNA accumulation, and systemic spread. Analyses of the BNYVV VSR mechanism of action led to the identification of the RNA-dependent RNA polymerase 6 (RDR6) pathway as a target of the virus VSR and the assignment of a VSR function to the ncRNA3.

Ins and Outs of Multipartite Positive-Strand RNA Plant Viruses: Packaging versus Systemic Spread.

Viruses possessing a non-segmented genome require a specific recognition of their nucleic acid to ensure its protection in a capsid. A similar feature exists for viruses having a segmented genome, usually consisting of viral genomic segments joined together into one viral entity. While this appears as a rule for animal viruses, the majority of segmented plant viruses package their genomic segments individually. To ensure a productive infection, all viral particles and thereby all segments have to be present in the same cell. Progression of the virus within the plant requires as well a concerted genome preservation to avoid loss of function. In this review, we will discuss the "life aspects" of chosen phytoviruses and argue for the existence of RNA-RNA interactions that drive the preservation of viral genome integrity while the virus progresses in the plant.

Rice stripe tenuivirus p2 may recruit or manipulate nucleolar functions through an interaction with fibrillarin to promote virus systemic movement.

Rice stripe virus (RSV) is the type species of the genus Tenuivirus and represents a major viral pathogen affecting rice production in East Asia. In this study, RSV p2 was fused to yellow fluorescent protein (p2-YFP) and expressed in epidermal cells of Nicotiana benthamiana. p2-YFP fluorescence was found to move to the nucleolus initially, but to leave the nucleolus for the cytoplasm forming numerous distinct bright spots there at later time points. A bimolecular fluorescence complementation (BiFC) assay showed that p2 interacted with fibrillarin and that the interaction occurred in the nucleus. Both the nucleolar localization and cytoplasmic distribution of p2-YFP fluorescence were affected in fibrillarin-silenced N. benthamiana. Fibrillarin depletion abolished the systemic movement of RSV, but not that of Tobacco mosaic virus (TMV) and Potato virus X (PVX). A Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) method was used to diminish RSV NS2 (encoding p2) or NS3 (encoding p3) during RSV infection. Silencing of NS3 alleviated symptom severity and reduced RSV accumulation, but had no obvious effects on virus movement and the timing of symptom development. However, silencing of NS2 abolished the systemic movement of RSV. The possibility that RSV p2 may recruit or manipulate nucleolar functions to promote virus systemic infection is discussed.

Fate of Pierce's Disease Strains of Xylella fastidiosa in Common Riparian Plants in California.

The fate of strains of the bacterium Xylella fastidiosa that cause Pierce's disease of grapevines was investigated in 33 species of mostly perennial plants common in riparian habitats in northern coastal California grape-growing regions. Plants were inoculated in the field with needle puncture using cultured cells of X. fastidiosa as inoculum or inoculated in the laboratory with infective insect vectors (Graphocephala atropunctata). Populations of X. fastidiosa were highest in most plant species within 3 to 6 weeks of inoculation, followed by declines in populations of viable bacteria over the next 3 to 4 months. Homogenates of petioles of California black walnut (Juglans hindsii) and coffeeberry (Rhamnus californica) inhibited in vitro growth of X. fas-tidiosa, precluding culture of the bacterium from these plants. Big leaf maple (Acer macrophyl-lum), California buckeye (Aesculus californica), California blackberry (Rubus ursinus), coast live oak (Quercus agrifolia), elderberry (Sambucus mexicana), French broom (Genista monspessulanus), periwinkle (Vinca major), valley oak (Quercus lobata), and the grape root-stock Vitis rupestris supported systemic populations of X. fastidiosa that survived throughout the year outdoors in Napa Valley, California.