Massive up-regulation of LBD transcription factors and EXPANSINs highlights the regulatory programs of rhizomania disease.

Rhizomania of sugar beet, caused by Beet necrotic yellow vein virus (BNYVV), is characterized by excessive lateral root (LR) formation leading to dramatic reduction of taproot weight and massive yield losses. LR formation represents a developmental process tightly controlled by auxin signaling through AUX/IAA-ARF responsive module and LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcriptional network. Several LBD transcription factors play central roles in auxin-regulated LR development and act upstream of EXPANSINS (EXPs), cell wall (CW)-loosening proteins involved in plant development via disruption of the extracellular matrix for CW relaxation and expansion. Here, we present evidence that BNYVV hijacks these auxin-regulated pathways resulting in formation LR and root hairs (RH). We identified an AUX/IAA protein (BvAUX28) as interacting with P25, a viral virulence factor. Mutational analysis indicated that P25 interacts with domains I and II of BvAUX28. Subcellular localization of co-expressed P25 and BvAUX28 showed that P25 inhibits BvAUX28 nuclear localization. Moreover, root-specific LBDs and EXPs were greatly upregulated during rhizomania development. Based on these data, we present a model in which BNYVV P25 protein mimics action of auxin by removing BvAUX28 transcriptional repressor, leading to activation of LBDs and EXPs. Thus, the evidence highlights two pathways operating in parallel and leading to uncontrolled formation of LRs and RHs, the main manifestation of the rhizomania syndrome.

Identification of a new PSII target site psbA mutation leading to D1 amino acid Leu218 Val exchange in the Chenopodium album D1 protein and comparison to cross-resistance profiles of known modifications at positions 251 and 264.

BACKGROUND: Resistance of Chenopodium album to triazinones and triazines can be caused by two amino acid exchanges, serine-264-glycine (Ser(264) Gly) and alanine-251-valine (Ala(251) Val), in the chloroplast D1 protein. This paper describes the identification of a biotype with a leucine-218-valine (Leu(218) Val) switch found in German sugar beet fields with unsatisfactory weed control. A greenhouse experiment has been performed to compare the resistance profile of the newly identified biotype with biotypes that carry the Ser(264) Gly and Ala(251) Val mutations. RESULTS: Application rate-response curves obtained from the greenhouse experiment showed that the Leu(218) Val exchange induced significant resistance against the triazinones but not against terbuthylazine. The level of resistance against the triazinones was higher in the Ser(264) Gly and Ala(251) Val biotypes compared with the Leu(218) Val biotype. All biotypes tested were more resistant to metribuzin than to metamitron. Following terbuthylazine treatment, Ser264 Gly displayed a high level of resistance, Ala(251) Val showed moderate resistance. A PCR-RFLP assay for Ser(264) Gly has been extended to include detection of Ala251 Val and Leu(218) Val mutations. CONCLUSION: The D1 Leu(218) Val substitution in C. album confers significant resistance to triazinones. This suggests that Leu(218) Val is involved in the binding of triazinones. First establishment of the resistance profiles of the three psbA mutations suggests that these mutations have been independently selected.

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.

A new molecular method for the rapid detection of a metamitron-resistant target site in Chenopodium album.

BACKGROUND: Resistance to photosystem II inhibitors-triazines (atrazine) and triazinones (metamitron, metribuzin)-in Chenopodium album L. is caused by the serine 264 to glycine mutation in the D1 protein. This mutation has been detected in C. album collections from Belgium with unsatisfactory metamitron efficacy in the field and was confirmed in greenhouse resistance bioassays. Incomplete herbicide efficacy in practice can also be caused by reduced uptake due to environmental conditions. Hence, for reliable differentiation and resistance identification, a rapid method for mutation detection in the target gene psbA is required. RESULTS: Dose-response curves obtained in herbicide greenhouse assays with metamitron-resistant and -susceptible reference biotypes showed that a dose of 2 L ha(-1) metamitron was suitable for discrimination. A psbA PCR-RFLP was developed, based on the presence of a FspBI restriction enzyme recognition site, covering D1 codon 264 in susceptible genotypes. A paper-based DNA extraction allowed direct processing of leaf samples already in the field. In order to detect the mutation even in mixed seed samples, a nested PCR-RFLP was also developed. CONCLUSION: The method allows exhaustive surveys screening C. album leaf or seed samples for the occurrence of the D1 Ser264Gly mutation to confirm or disprove metamitron resistance in the case of unsatisfactory control.

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 factor's 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.

Functional mapping of PVX RNA-dependent RNA-replicase using pentapeptide scanning mutagenesis-Identification of regions essential for replication and subgenomic RNA amplification.

The replicase protein of Potato virus X (PVX), type species of the genus Potexvirus, was selected to identify regions essential for replication and subgenomic RNA synthesis. Replicase amino acid (aa) sequence alignment of 16 Potexvirus species resulted in the detection of overall sequence homology of 34.4-65.4%. Two regions of consensus with a high proportion of conserved aa (1-411 and 617-1437 according to PVX) were separated by a hyper-variable linker region. Pentapeptide scanning (PS) mutagenesis in a PVX full-length clone expressing green fluorescent protein (GFP) was carried out. For 69 selected PS-mutants where insertions were spread randomly over the replicase ORF the position of the insertion was determined. The replication activity was evaluated by GFP expression from subgenomic viral RNA of PVX replicase mutants. Only one functional PS-mutant was detected in the N-terminal 430 aa, containing the conserved methyltransferase domain of the protein. In the linker region from aa 430-595, nine mutations were discovered which did not induce significant effects on the replicase replication ability. The part of the protein including helicase and polymerase domains was highly intolerant for the PS insertion as demonstrated by 24 independent more or less uniformly spread mutants.