Mark Varrelmann

Mutations in the coat protein gene of Plum pox virus suppress particle assembly, heterologous encapsidation and complementation in transgenic plants of Nicotiana benthamiana

Two different motifs in the coat protein (CP) of Plum pox virus (PPV) (R(3015)Q(3016), D(3059)) were mutated by replacing the respective amino acids with others possessing different chemical properties. The mutated CP genes were introduced into an infectious full-length clone of PPV (p35PPV-NAT) to investigate their influence on systemic infection of transgenic wild-type PPV CP-expressing and non-transgenic plants of Nicotiana benthamiana. All mutants failed to establish systemic infections in non-transgenic N. benthamiana plants, but were complemented by intact CP in transgenic plants. Moreover, the CP-RQ-D mutant (carrying mutations in both the RQ and D motifs) was introduced into p35PPV-NAT engineered to express beta-glucuronidase (GUS) for direct observation of systemic movement and particle assembly in N. benthamiana leaves. GUS-staining revealed that the CP mutant (RQ-D) was restricted to initially infected cells without forming virions. Systemic movement and particle assembly were restored in CP-transgenic N. benthamiana plants. Finally, transgenic N. benthamiana plants were generated that expressed each of the three mutated CP genes. Homozygous T(2) lines were selected and tested for resistance to PPV. Immunogold labelling and electron microscopy revealed that heterologous encapsidation with challenging Chilli veinal mottle virus and Potato virus Y was suppressed in these lines. In addition, assembly mutants did not complement CP-defective p35PPV-NAT. The possible use of modified viral CP genes for the production of virus-resistant transgenic plants, thereby reducing the putative risks of heterologous encapsidation and complementation, is discussed.

Chickpea chlorotic stunt virus: A New Polerovirus Infecting Cool-Season Food Legumes in Ethiopia

ABSTRACT Serological analysis of diseased chickpea and faba bean plantings with yellowing and stunting symptoms suggested the occurrence of an unknown or uncommon member of the family Luteoviridae in Ethiopia. Degenerate primers were used for reverse transcriptase-polymerase chain reaction amplification of the viral coat protein (CP) coding region from both chickpea and faba bean samples. Cloning and sequencing of the amplicons yielded nearly identical (96%) nucleotide sequences of a previously unrecognized species of the family Luteoviridae, with a CP amino acid sequence most closely related (identity of approximately 78%) to that of Groundnut rosette assistor virus. The complete genome (5,900 nts) of a faba bean isolate comprised six major open reading frames characteristic of polero-viruses. Of the four aphid species tested, only Aphis craccivora transmitted the virus in a persistent manner. The host range of the virus was confined to a few species of the family Fabaceae. A rabbit antiserum raised against virion preparations cross-reacted unexpectedly with Beet western yellows virus-like viruses. This necessitated the production of murine monoclonal antibodies which, in combination with the polyclonal antiserum, permitted both sensitive and specific detection of the virus in field samples by triple-antibody sandwich, enzyme-linked immunosorbent assay. Because of the characteristic field and greenhouse symptoms in chickpea, the name Chickpea chlorotic stunt virus is proposed for this new member of the genus Polerovirus (family Luteoviridae).

Functional characterization and subcellular localization of the 16K cysteine-rich suppressor of gene silencing protein of tobacco rattle virus.

The 16 kDa cysteine-rich protein (16K) of tobacco rattle virus (TRV) is known to partially suppress RNA silencing in Drosophila cells. In this study, we show that 16K suppresses RNA silencing in green fluorescent protein (GFP)-transgenic Nicotiana benthamiana plants using an Agrobacterium-mediated transient assay. 16K slightly reduced the accumulation of short interfering RNAs (siRNA) of GFP, suggesting that the protein may interfere with the initiation and/or maintenance of RNA silencing. Deletion of either the N- or C-terminal part of 16K indicated that the entire 16K open reading frame (ORF) is necessary for its silencing suppression function. Pentapeptide insertion scanning mutagenesis (PSM) revealed that only two short regions of 16K tolerated five extra amino acid insertions without considerable reduction in its silencing suppression function. The tolerant regions coincide with sequence variability between tobravirus cysteine-rich proteins, indicating a strong functional and/or structural conservation of TRV 16K. Confocal laser scanning microscopy of transiently expressed 16K fusions to red fluorescent protein (RFP) revealed a predominant cytoplasmic localization and, in addition, a nuclear localization. In contrast, fusions of RFP with the N-terminal region of 16K localized exclusively to the cytoplasm, whereas fusions between RFP and the C-terminal region of 16K displayed an exclusive nuclear localization. Further analysis of 16K-derived peptide fusions demonstrated that the 16K C-terminal region contained at least two functional bipartite nuclear localization signals which were independently capable of nuclear targeting.

Transgenic or Plant Expression Vector-Mediated Recombination of Plum Pox Virus

ABSTRACT Different mutants of an infectious full-length clone (p35PPV-NAT) of Plum pox virus (PPV) were constructed: three mutants with mutations of the assembly motifs RQ and DF in the coat protein gene (CP) and two CP chimeras with exchanges in the CP core region ofZucchini yellow mosaic virus and Potato virus Y. The assembly mutants were restricted to single infected cells, whereas the PPV chimeras were able to produce systemic infections inNicotiana benthamiana plants. After passages in different transgenic N. benthamiana plants expressing the PPV CP gene with a complete (plant line 4.30.45.) or partially deleted 3′-nontranslated region (3′-NTR) (plant line 17.27.4.), characterization of the viral progeny of all mutants revealed restoration of wild-type virus by recombination with the transgenic CP RNA only in the presence of the complete 3′-NTR (4.30.45.). Reconstitution of wild-type virus was also observed following cobombardment of different assembly-defective p35PPV-NAT together with a movement-defective plant expression vector of Potato virus X expressing the intact PPV-NAT CP gene transiently in nontransgenic N. benthamiana plants. Finally, a chimeric recombinant virus was detected after cobombardment of defective p35PPV-NAT with a plant expression vector-derived CP gene from the sour cherry isolate of PPV (PPV-SoC). This chimeric virus has been established by a double recombination event between the CP-defective PPV mutant and the intact PPV-SoC CP gene. These results demonstrate that viral sequences can be tested for recombination events without the necessity for producing transgenic plants.

Tobacco rattle virus 29K Movement Protein Is the Elicitor of Extreme and Hypersensitive-like Resistance in Two Cultivars of Solanum tuberosum

Leaf infection experiments were used to analyze the host responses of Solanum tuberosum cultivars known to be resistant or susceptible to natural, nematode-mediated infection of tubers and necrosis induction (spraing) by Tobacco rattle virus (TRV) isolate PpK20 (TRV-PpK20). Extreme and hypersensitive-like resistance (ER and HR-like, respectively) as well as spreading veinal necrosis and systemic infection were observed. Agroinfection of leaves with a DsRed-expressing TRV cDNA clone revealed ER to function on the single-cell level, inhibiting virus replication and possessing the potential to initiate a cell death response. HR-like necrosis was characterized by initial virus replication and cell-to-cell movement before the onset of necrosis. Transient agroexpression and Potato virus X (PVX)-mediated expression assays demonstrated that the 29K-PpK20 movement protein (MP) can elicit ER and HR-like cell-death. A TRV isolate, PpO85M, known to overcome the resistance to spraing in plants that are resistant to TRV-PpK20 encoded a variant 29K protein which did not elicit HR in PpK20-HR plants. Our results show that the TRV MP is the elicitor of both ER and HR-like cell-death, that no other TRV-encoded proteins or RNA replication are required for its elicitor activity, and that the host reactions are likely to be controlled by single dominant resistance genes.

Identification of Beet necrotic yellow vein virus P25 Pathogenicity Factor–Interacting Sugar Beet Proteins That Represent Putative Virus Targets or Components of Plant Resistance

Beet necrotic yellow vein virus (BNYVV) induces the most important disease threatening sugar beet. The growth of partially resistant hybrids carrying monogenic dominant resistance genes stabilize yield but are unable to entirely prevent virus infection and replication. P25 is responsible for symptom development and previous studies have shown that recently occurring resistance-breaking isolates possess increased P25 variability. To better understand the viral pathogenicity factors interplay with plant proteins and to possibly unravel the molecular basis of sugar beet antivirus resistance, P25 was applied in a yeast two-hybrid screen of a resistant sugar beet cDNA library. This screen identified candidate proteins recognized as orthologues from other plant species which are known to be expressed following pathogen infection and involved in plant defense response. Most of the candidates potentially related to host-pathogen interactions were involved in the ubiquitylation process and plants response to stress, and were part of cell and metabolism components. The interaction of several candidate genes with P25 was confirmed in Nicotiana benthamiana leaf cells by transient agrobacterium-mediated expression applying bimolecular fluorescence complementation assay. The putative functions of several of the candidates identified support previous findings and present first targets for understanding the BNYVV pathogenicity and antivirus resistance mechanism.

Molecular, serological and biological variation among chickpea chlorotic stunt virus isolates from five countries of North Africa and West Asia.

Chickpea chlorotic stunt virus (CpCSV), a proposed new member of the genus Polerovirus (family Luteoviridae), has been reported only from Ethiopia. In attempts to determine the geographical distribution and variability of CpCSV, a pair of degenerate primers derived from conserved domains of the luteovirus coat protein (CP) gene was used for RT-PCR analysis of various legume samples originating from five countries and containing unidentified luteoviruses. Sequencing of the amplicons provided evidence for the occurrence of CpCSV also in Egypt, Morocco, Sudan, and Syria. Phylogenetic analysis of the CP nucleotide sequences of 18 samples from the five countries revealed the existence of two geographic groups of CpCSV isolates differing in CP sequences by 8–10%. Group I included isolates from Ethiopia and Sudan, while group II comprised those from Egypt, Morocco and Syria. For distinguishing these two groups, a simple RFLP test using HindIII and/or PvuII for cleavage of CP-gene-derived PCR products was developed. In ELISA and immunoelectron microscopy, however, isolates from these two groups could not be distinguished with rabbit antisera raised against a group-I isolate from Ethiopia (CpCSV-Eth) and a group-II isolate from Syria (CpCSV-Sy). Since none of the ten monoclonal antibodies (MAbs) that had been produced earlier against CpCSV-Eth reacted with group-II isolates, further MAbs were produced. Of the seven MAbs raised against CpCSV-Sy, two reacted only with CpCSV-Sy and two others with both CpCSV-Sy and -Eth. This indicated that there are group I- and II-specific and common (species-specific) epitopes on the CpCSV CP and that the corresponding MAbs are suitable for specific detection and discrimination of CpCSV isolates. Moreover, CpCSV-Sy (group II) caused more severe stunting and yellowing in faba bean than CpCSV-Eth (group I). In conclusion, our data indicate the existence of a geographically associated variation in the molecular, serological and presumably biological properties of CpCSV.

The P25 pathogenicity factor of Beet necrotic yellow vein virus targets the sugar beet 26S proteasome involved in the induction of a hypersensitive resistance response via interaction with an F-box protein.

P25, a Beet necrotic yellow vein virus (BNYVV) pathogenicity factor, interacts with a sugar beet protein with high homology to Arabidopsis thaliana kelch repeat containing F-box family proteins (FBK) of unknown function in yeast. FBK are members of the Skp1-Cullin-F-box (SCF) complex that mediate protein degradation. Here, we confirm this sugar beet FBK-P25 interaction in vivo and in vitro and provide evidence for in planta interaction and similar subcellular distribution in Nicotiana tabacum leaf cells. P25 even interacts with an FBK from A. thaliana, a BNYVV nonhost. FBK functional classification was possible by demonstrating the interaction with A. thaliana orthologs of Skp1-like (ASK) genes, a member of the SCF E3 ligase. By means of a yeast two-hybrid bridging assay, a direct effect of P25 on SCF-complex formation involving ASK1 protein was demonstrated. FBK transient Agrobacterium tumefaciens-mediated expression in N. benthamiana leaves induced a hypersensitive response. The full-length F-box protein consists of one F-box domain followed by two kelch repeats, which alone were unable to interact with P25 in yeast and did not lead to cell-death induction. The results support the idea that P25 is involved in virus pathogenicity in sugar beet and suggest suppression of resistance response.

Evidence for similarity-assisted recombination and predicted stem-loop structure determinant in potato virus X RNA recombination

Virus RNA recombination, one of the main factors for genetic variability and evolution, is thought to be based on different mechanisms. Here, the recently described in vivo potato virus X (PVX) recombination assay [Draghici, H.-K. & Varrelmann, M. (2009). J Virol 83, 7761-7769] was applied to characterize structural parameters of recombination. The assay uses an Agrobacterium-mediated expression system incorporating a PVX green fluorescent protein (GFP)-labelled full-length clone. The clone contains a partial coat protein (CP) deletion that causes defectiveness in cell-to-cell movement, together with a functional CP+3 non-translated region (ntr) transcript, in Nicotiana benthamiana leaf tissue. The structural parameters assessed were the length of sequence overlap, the distance between mutations and the degree of sequence similarity. The effects on the observed frequency of reconstitution and the composition of the recombination products were characterized. Application of four different type X intact PVX CP genes with variable composition allowed the estimation of the junction sites of precise homologous recombination. Although one template switch would have been sufficient for functional reconstitution, between one and seven template switches were observed. Use of PVX-GFP mutants with CP deletions of variable length resulted in a linear decrease of the reconstitution frequency. The critical length observed for homologous recombination was 20-50 nt. Reduction of the reconstitution frequency was obtained when a phylogenetically distant PVX type Bi CP gene was used. Finally, the prediction of CP and 3-ntr RNA secondary structure demonstrated that recombination-junction sites were located mainly in regions of stem-loop structures, allowing the recombination observed to be categorized as similarity-assisted.

Effect of sugar beet genotype on the Beet necrotic yellow vein virus P25 pathogenicity factor and evidence for a fitness penalty in resistance‐breaking strains

Beet necrotic yellow vein virus (BNYVV), vectored by Polymyxa betae, causes rhizomania in sugar beet. For disease control, the cultivation of hybrids carrying Rz1 resistance is crucial, but is compromised by resistance-breaking (RB) strains with specific mutations in the P25 protein at amino acids 67-70 (tetrad). To obtain evidence for P25 variability from soil-borne populations, where the virus persists for decades, populations with wild-type (WT) and RB properties were analysed by P25 deep sequencing. The level of P25 variation in the populations analysed did not correlate with RB properties. Remarkably, one WT population contained P25 with RB mutations at a frequency of 11%. To demonstrate selection by Rz1 and the influence of RB mutations on relative fitness, competition experiments between strains were performed. Following a mixture of strains with four RNAs, a shift in tetrad variants was observed, suggesting that strains did not mix or transreplicate. The plant genotype exerted a clear influence on the frequency of RB tetrads. In Rz1 plants, the RB variants outcompeted the WT variants, and mostly vice versa in susceptible plants, demonstrating a relative fitness penalty of RB mutations. The strong genotype effect supports the hypothesized Rz1u2005RB strain selection with four RNAs, suggesting that a certain tetrad needs to become dominant in a population to influence its properties. Tetrad selection was not observed when an RB strain, with an additional P26 protein encoded by a fifth RNA, competed with a WT strain, supporting its role as a second BNYVV pathogenicity factor and suggesting the reassortment of both types.