Putative NAD(P)-Binding Rossmann Fold Protein Is Involved in Chitosan-Induced Peroxidase Activity and Lipoxygenase Expression in Physcomitrium patens.

Oxidative burst, the rapid production of high levels of reactive oxygen species in response to external stimuli, is an early defense reaction against pathogens. The fungal elicitor chitosan causes an oxidative burst in the moss Physcomitrium patens (formerly Physcomitrella patens), mainly due to the peroxidase enzyme Prx34. To better understand the chitosan responses in P. patens, we conducted a screen of part of a P. patens mutant collection to isolate plants with less peroxidase activity than wild-type (WT) plants after chitosan treatment. We isolated a P. patens mutant that affected the gene encoding NAD(P)-binding Rossmann fold protein (hereafter, Rossmann fold protein). Three Rossmann fold protein-knockout (KO) plants (named Rossmann fold KO lines) were generated and used to assess extracellular peroxidase activity and expression of defense-responsive genes, including alternative oxidase, lipoxygenase (LOX), NADPH oxidase, and peroxidase (Prx34) in response to chitosan treatment. Extracellular (apoplastic) peroxidase activity was significantly lower in Rossmann fold KO lines than in WT plants after chitosan treatments. Expression of the LOX gene in Rossmann fold KO plants was significantly lower before and after chitosan treatment when compared with WT. Peroxidase activity assays together with gene expression analyses suggest that the Rossmann fold protein might be an important component of the signaling pathway leading to oxidative burst and basal expression of the LOX gene in P. patens. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

In Vitro Identification and In Vivo Confirmation of Inhibitors for Sweet Potato Chlorotic Stunt Virus RNA Silencing Suppressor, a Viral RNase III.

Sweet potato virus disease (SPVD), caused by synergistic infection of Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato feathery mottle virus (SPFMV), is responsible for substantial yield losses all over the world. However, there are currently no approved treatments for this severe disease. The crucial role played by RNase III of SPCSV (CSR3) as an RNA silencing suppressor during the viruses' synergistic interaction in sweetpotato makes it an ideal drug target for developing antiviral treatment. In this study, high-throughput screening (HTS) of small molecular libraries targeting CSR3 was initiated by a virtual screen using Glide docking, allowing the selection of 6,400 compounds out of 136,353. We subsequently developed and carried out kinetic-based HTS using fluorescence resonance energy transfer technology, which isolated 112 compounds. These compounds were validated with dose-response assays including kinetic-based HTS and binding affinity assays using surface plasmon resonance and microscale thermophoresis. Finally, the interference of the selected compounds with viral accumulation was verified in planta In summary, we identified five compounds belonging to two structural classes that inhibited CSR3 activity and reduced viral accumulation in plants. These results provide the foundation for developing antiviral agents targeting CSR3 to provide new strategies for controlling sweetpotato virus diseases.IMPORTANCE We report here a high-throughput inhibitor identification method that targets a severe sweetpotato virus disease caused by coinfection with two viruses (SPCSV and SPFMV). The disease is responsible for up to 90% yield losses. Specifically, we targeted the RNase III enzyme encoded by SPCSV, which plays an important role in suppressing the RNA silencing defense system of sweetpotato plants. Based on virtual screening, laboratory assays, and confirmation in planta, we identified five compounds that could be used to develop antiviral drugs to combat the most severe sweetpotato virus disease.

Viral suppressor of RNA silencing in vascular plants also interferes with the development of the bryophyte Physcomitrella patens.

Plant viruses are important pathogens able to overcome plant defense mechanisms using their viral suppressors of RNA silencing (VSR). Small RNA pathways of bryophytes and vascular plants have significant similarities, but little is known about how viruses interact with mosses. This study elucidated the responses of Physcomitrella patens to two different VSRs. We transformed P. patens plants to express VSR P19 from tomato bushy stunt virus and VSR 2b from cucumber mosaic virus, respectively. RNA sequencing and quantitative PCR were used to detect the effects of VSRs on gene expression. Small RNA (sRNA) sequencing was used to estimate the influences of VSRs on the sRNA pool of P. patens. Expression of either VSR-encoding gene caused developmental disorders in P. patens. The transcripts of four different transcription factors (AP2/erf, EREB-11 and two MYBs) accumulated in the P19 lines. sRNA sequencing revealed that VSR P19 significantly changed the microRNA pool in P. patens. Our results suggest that VSR P19 is functional in P. patens and affects the abundance of specific microRNAs interfering with gene expression. The results open new opportunities for using Physcomitrella as an alternative system to study plant-virus interactions.

Residues R192 and K225 in RNA-Binding Pocket of Tobacco Vein Banding Mosaic Virus CP Control Virus Cell-to-Cell Movement and Replication.

Potyviruses move to neighboring cells in the form of virus particles or a coat protein (CP)-containing ribonucleoprotein complex. However, the precise roles of RNA-binding residues in potyviral CP in viral cell-to-cell movement remain to be elucidated. In this study, we predicted the three-dimensional model of tobacco vein banding mosaic virus (TVBMV)-encoded CP and found nine residues presumably located in the CP RNA-binding pocket. Substitutions of the two basic residues at positions 192 and 225 (R192 and K225) with either alanine, cysteine, or glutamic acid abolished TVBMV cell-to-cell and systemic movement in Nicotiana benthamiana plants. These substitutions also reduced the replication of the mutant viruses. Results from the electrophoretic mobility shift assay showed that the RNA-binding activity of mutant CPs derived from R192 or K225 substitutions was significantly lower than that of wild-type CP. Analysis of purified virus particles showed that mutant viruses with R192 or K225 substitutions formed RNA-free virus-like particles. Mutations of R192 and K225 did not change the CP plasmodesmata localization. The wild-type TVBMV CP could rescue the deficient cell-to-cell movement of mutant viruses. Moreover, deletion of any of the other seven residues also abolished TVBMV cell-to-cell movement and reduced the CP RNA-binding activity. The corresponding nine residues in watermelon mosaic virus CP were also found to play essential roles in virus cell-to-cell movement. In conclusion, residues R192 and K225 in the CP RNA-binding pocket are critical for viral RNA binding and affect both virus replication and cell-to-cell movement.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Next-Generation Sequencing-Based Detection of Common Bean Viruses in Wild Plants from Tanzania and Their Mechanical Transmission to Common Bean Plants.

Viral diseases are a major threat for common bean production. According to recent surveys, >15 different viruses belonging to 11 genera were shown to infect common bean (Phaseolus vulgaris L.) in Tanzania. Virus management requires an understanding of how viruses survive from one season to the next. During this study, we explored the possibility that alternative host plants have a central role in the survival of common bean viruses. We used next-generation sequencing (NGS) techniques to sequence virus-derived small interfering RNAs together with conventional reverse-transcription PCRs (RT-PCRs) to detect viruses in wild plants. Leaf samples for RNA extraction and NGS were collected from 1,430 wild plants around and within common bean fields in four agricultural zones in Tanzania. At least partial genome sequences of viruses potentially belonging to 25 genera were detected. The greatest virus diversity was detected in the eastern and northern zones, whereas wild plants in the Lake zone and especially in the southern highlands zone showed only a few viruses. The RT-PCR analysis of all collected plant samples confirmed the presence of yam bean mosaic virus and peanut mottle virus in wild legume plants. Of all viruses detected, only two viruses, cucumber mosaic virus and a novel bromovirus related to cowpea chlorotic mottle virus and brome mosaic virus, were mechanically transmitted from wild plants to common bean plants. The data generated during this study are crucial for the development of viral disease management strategies and predicting crop viral disease outbreaks in different agricultural regions in Tanzania and beyond.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Nuclear proteome of virus-infected and healthy potato leaves.

BACKGROUND: Infection of plants by viruses interferes with expression and subcellular localization of plant proteins. Potyviruses comprise the largest and most economically damaging group of plant-infecting RNA viruses. In virus-infected cells, at least two potyviral proteins localize to nucleus but reasons remain partly unknown. RESULTS: In this study, we examined changes in the nuclear proteome of leaf cells from a diploid potato line (Solanum tuberosum L.) after infection with potato virus A (PVA; genus Potyvirus; Potyviridae) and compared the data with that acquired for healthy leaves. Gel-free liquid chromatography-coupled to tandem mass spectrometry was used to identify 807 nuclear proteins in the potato line v2-108; of these proteins, 370 were detected in at least two samples of healthy leaves. A total of 313 proteins were common in at least two samples of healthy and PVA-infected leaves; of these proteins, 8 showed differential accumulation. Sixteen proteins were detected exclusively in the samples from PVA-infected leaves, whereas other 16 proteins were unique to healthy leaves. The protein Dnajc14 was only detected in healthy leaves, whereas different ribosomal proteins, ribosome-biogenesis proteins, and RNA splicing-related proteins were over-represented in the nuclei of PVA-infected leaves. Two virus-encoded proteins were identified in the samples of PVA-infected leaves. CONCLUSIONS: Our results show that PVA infection alters especially ribosomes and splicing-related proteins in the nucleus of potato leaves. The data increase our understanding of potyvirus infection and the role of nucleus in infection. To our knowledge, this is the first study of the nuclear proteome of potato leaves and one of the few studies of changes occurring in nuclear proteomes in response to plant virus infection.

Differential Requirement of the Ribosomal Protein S6 and Ribosomal Protein S6 Kinase for Plant-Virus Accumulation and Interaction of S6 Kinase with Potyviral VPg.

Ribosomal protein S6 (RPS6) is an indispensable plant protein regulated, in part, by ribosomal protein S6 kinase (S6K) which, in turn, is a key regulator of plant responses to stresses and developmental cues. Increased expression of RPS6 was detected in Nicotiana benthamiana during infection by diverse plant viruses. Silencing of the RPS6 and S6K genes in N. benthamiana affected accumulation of Cucumber mosaic virus, Turnip mosaic virus (TuMV), and Potato virus A (PVA) in contrast to Turnip crinkle virus and Tobacco mosaic virus. In addition, the viral genome-linked protein (VPg) of TuMV and PVA interacted with S6K in plant cells, as detected by bimolecular fluorescence complementation assay. The VPg-S6K interaction was detected in cytoplasm, nucleus, and nucleolus, whereas the green fluorescent protein-tagged S6K alone showed cytoplasmic localization only. These results demonstrate that the requirement for RPS6 and S6K differs for diverse plant viruses with different translation initiation strategies and suggest that potyviral VPg-S6K interaction may affect S6K functions in both the cytoplasm and the nucleus.

Suppression of RNAi by dsRNA-degrading RNaseIII enzymes of viruses in animals and plants.

Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)--mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in unrelated RNA and DNA viruses and can be detected in viral genomes using gene modeling and protein structure prediction programs.

Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens.

In seed plants, strigolactones (SLs) regulate architecture and induce mycorrhizal symbiosis in response to environmental cues. SLs are formed by combined activity of the carotenoid cleavage dioxygenases (CCDs) 7 and 8 from 9-cis-ß-carotene, leading to carlactone that is converted by cytochromes P450 (clade 711; MAX1 in Arabidopsis) into various SLs. As Physcomitrella patens possesses CCD7 and CCD8 homologs but lacks MAX1, we investigated if PpCCD7 together with PpCCD8 form carlactone and how deletion of these enzymes influences growth and interactions with the environment. We investigated the enzymatic activity of PpCCD7 and PpCCD8 in vitro, identified the formed products by high performance liquid chromatography (HPLC) and LC-MS, and generated and analysed ΔCCD7 and ΔCCD8 mutants. We defined enzymatic activity of PpCCD7 as a stereospecific 9-cis-CCD and PpCCD8 as a carlactone synthase. ΔCCD7 and ΔCCD8 lines showed enhanced caulonema growth, which was revertible by adding the SL analogue GR24 or carlactone. Wild-type (WT) exudates induced seed germination in Orobanche ramosa. This activity was increased upon phosphate starvation and abolished in exudates of both mutants. Furthermore, both mutants showed increased susceptibility to phytopathogenic fungi. Our study reveals the deep evolutionary conservation of SL biosynthesis, SL function, and its regulation by biotic and abiotic cues.

Graphical genotyping as a method to map Ny (o,n)sto and Gpa5 using a reference panel of tetraploid potato cultivars.

KEY MESSAGE: The method of graphical genotyping is applied to a panel of tetraploid potato cultivars to visualize haplotype sharing. The method allowed to map genes involved in virus and nematode resistance. The physical coordinates of the amount of linkage drag surrounding these genes are easily interpretable. Graphical genotyping is a visually attractive and easily interpretable method to represent genetic marker data. In this paper, the method is extended from diploids to a panel of tetraploid potato cultivars. Application of filters to select a subset of SNPs allows one to visualize haplotype sharing between individuals that also share a specific locus. The method is illustrated with cultivars resistant to Potato virus Y (PVY), while simultaneously selecting for the absence of the SNPs in susceptible clones. SNP data will then merge into an image which displays the coordinates of a distal genomic region on the northern arm of chromosome 11 where a specific haplotype is introgressed from the wild potato species S. stoloniferum (CPC 2093) carrying a gene (Ny (o,n)sto ) conferring resistance to two PVY strains, PVYO and PVYNTN. Graphical genotyping was also successful in showing the haplotypes on chromosome 12 carrying Ry-f sto , another resistance gene derived from S. stoloniferum conferring broad-spectrum resistance to PVY, as well as chromosome 5 haplotypes from S. vernei, with the Gpa5 locus involved in resistance against Globodera pallida cyst nematodes. The image also shows shortening of linkage drag by meiotic recombination of the introgression segment in more recent breeding material. Identity-by-descent was found to be a requirement for using graphical genotyping, which is proposed as a non-statistical alternative method for gene discovery, as compared with genome-wide association studies. The potential and limitations of the method are discussed.

Repeated Applications of a Nonpathogenic Streptomyces Strain Enhance Development of Suppressiveness to Potato Common Scab.

Potato common scab caused by several Streptomyces spp. is an important disease with no effective methods of control. Suppressiveness against common scab can develop in soil as a result of long-term potato monoculture and has been associated with nonpathogenic Streptomyces spp. To determine whether the development of scab suppressiveness could be enhanced, the effect of repeated applications of an antagonistic Streptomyces strain on common scab was investigated in a long-term field trial over 5 years. Streptomyces strain 272 applied annually at planting consistently suppressed development of common scab symptoms. On scab-susceptible potato cultivar Bintje, strain 272 reduced disease severity, on average, by 43%; whereas, on the scab-tolerant Nicola, the strain reduced both disease incidence and severity by 43 and 59%, respectively. Regardless of disease pressure, the combined use of strain 272 and the tolerant cultivar reduced the scab coverage to a negligible level. After a single application of strain 272, efficient disease suppression did not persist in the soil to the following growing season. However, when strain 272 was applied in three or more consecutive years, the soil remained suppressive to scab for at least 2 years beyond the last application, suggesting that, with repeated applications, it may be possible to enhance development of scab suppression in soil.

Diagnosis and discovery of fungal viruses using deep sequencing of small RNAs.

Analysis of virus-derived small RNAs with high-throughput sequencing has been successful for detecting novel viruses in plants and invertebrates. However, the applicability of this method has not been demonstrated in fungi, although fungi were among the first organisms reported to utilize RNA silencing. Here, we used virus-infected isolates of the fungal species complex Heterobasidion annosum sensu lato as a model system to test whether mycovirus genome segments can be detected with small RNA deep sequencing. Species of the genus Heterobasidion are some of the most devastating forest pathogens in boreal forests. These fungi cause wood decay and are commonly infected with species of the family Partitiviridae and the unassigned virus species Heterobasidion RNA virus 6. Small RNA deep sequencing allowed the simultaneous detection of all eight double-stranded RNA virus strains known to be present in the tested samples and one putative mitovirus species (family Narnaviridae) with a single-stranded RNA genome, designated here as Heterobasidion mitovirus 1. Prior to this study, no members of the family Narnaviridae had been described as infecting species of Heterobasidion. Quantification of viral double- and single-stranded RNA with quantitative PCR indicated that co-infecting viral species and viruses with segmented genomes can be detected with small RNA deep sequencing despite vast differences in the amount of RNA. This is the first study demonstrating the usefulness of this method for detecting fungal viruses. Moreover, the results suggest that viral genomes are processed into small RNAs by different species of Heterobasidion.

Diversity and evolution of potato mop-top virus.

Nearly complete sequences of RNA-CP and 3'-proximal RNA-TGB were determined for 43 samples of potato mop-top virus (PMTV) originating from potato tubers and field soil from Sweden, Denmark and the USA. The results showed limited diversity and no strict geographical grouping, suggesting only a few original introductions of PMTV from the Andes. Two distinguishable types of RNA-CP and RNA-TGB were found in the samples, but no specific combination of them correlated with spraing symptoms in tubers. Lack of positive selection in the coding sequences indicates that there is no specific molecular adaptation of PMTV to new vectors or hosts.

Elucidation of virus-host interactions to enhance resistance breeding for control of virus diseases in potato.

Potato virus Y (PVY) and Potato mop-top virus (PMTV) are viruses whose geographical distribution is expanding and economic losses are increasing, in contrast to most of other viruses infecting potato crops. Most potato cultivars lack broad-spectrum resistance to the new, genetically complex strains of PVY, and no efficient resistance to PMTV is known in potato. Control of the vectors of these viruses is not an efficient or possible strategy to prevent infections. Studies on molecular virus-host interactions can discover plant genes that are important to viral infection or antiviral defence. Both types of genes may be utilized in resistance breeding, which is discussed in this paper. The advanced gene technologies provide means to fortify potato cultivars with effective virus resistance genes or mutated, non-functional host factors that interfere with virus infection.

Protein secretome of moss plants (Physcomitrella patens) with emphasis on changes induced by a fungal elicitor.

Studies on extracellular proteins (ECPs) contribute to understanding of the multifunctional nature of apoplast. Unlike vascular plants (tracheophytes), little information about ECPs is available from nonvascular plants, such as mosses (bryophytes). In this study, moss plants (Physcomitrella patens) were grown in liquid culture and treated with chitosan, a water-soluble form of chitin that occurs in cell walls of fungi and insects and elicits pathogen defense in plants. ECPs released to the culture medium were compared between chitosan-treated and nontreated control cultures using quantitative mass spectrometry (Orbitrap) and 2-DE-LC-MS/MS. Over 400 secreted proteins were detected, of which 70% were homologous to ECPs reported in tracheophyte secretomes. Bioinformatics analyses using SignalP and SecretomeP predicted classical signal peptides for secretion (37%) or leaderless secretion (27%) for most ECPs of P. patens, but secretion of the remaining proteins (36%) could not be predicted using bioinformatics. Cultures treated with chitosan contained 72 proteins not found in untreated controls, whereas 27 proteins found in controls were not detected in chitosan-treated cultures. Pathogen defense-related proteins dominated in the secretome of P. patens, as reported in tracheophytes. These results advance knowledge on protein secretomes of plants by providing a comprehensive account of ECPs of a bryophyte.

Silencing suppressor protein VPg of a potyvirus interacts with the plant silencing-related protein SGS3.

Viral genome-linked protein (VPg) of potyviruses is involved in multiple steps of the potyvirus infection cycle, including viral multiplication and movement in plants. Recently, we showed that VPg of Potato virus A (PVA; genus Potyvirus) suppresses sense-mediated RNA silencing, which is linked to one or both nuclear or nucleolar localization. Here, we studied interactions between VPg and components of the plant RNA silencing pathway. Results showed that VPg interacts with the SGS3 protein of Solanum tuberosum and Arabidopsis thaliana, as shown by yeast two-hybrid analysis and bimolecular fluorescence complementation assays. VPg-SGS3 interactions co-localized with small cytoplasmic bodies that contained plant RNA-dependent RNA polymerase 6 (RDR6) (likely SGS3/RDR6 bodies). The N-terminal zinc finger (ZF) domain of SGS3 was the main determinant of the VPg interaction. Our data also suggest that the ZF domain controls SGS3 localization. SGS3 homodimerization was controlled by multiple protein regions. The VPg-SGS3 interaction appeared beneficial for PVA, as viral RNA levels correlated positively with sgs3 mRNA levels in the SGS3-silenced and SGS3-overexpressing leaves of Nicotiana benthamiana. The data support the idea that VPg acts as a suppressor of RNA silencing and suggest that an interaction with SGS3 may be important, especially in suppression of sense-mediated RNA silencing.

Binding and processing of small dsRNA molecules by the class 1 RNase III protein encoded by sweet potato chlorotic stunt virus.

Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) causes heavy yield losses in sweet potato plants co-infected with other viruses. The dsRNA-specific class 1 RNase III-like endoribonuclease (RNase3) encoded by SPCSV suppresses post-transcriptional gene silencing and eliminates antiviral defence in sweet potato plants in an endoribonuclease activity-dependent manner. RNase3 can cleave long dsRNA molecules, synthetic small interfering RNAs (siRNAs), and plant- and virus-derived siRNAs extracted from sweet potato plants. In this study, conditions for efficient expression and purification of enzymically active recombinant RNase3 were established. Similar to bacterial class 1 RNase III enzymes, RNase3-Ala (a dsRNA cleavage-deficient mutant) bound to and processed double-stranded siRNA (ds-siRNA) as a dimer. The results support the classification of SPCSV RNase3 as a class 1 RNase III enzyme. There is little information about the specificity of RNase III enzymes on small dsRNAs. In vitro assays indicated that ds-siRNAs and microRNAs (miRNAs) with a regular A-form conformation were cleaved by RNase3, but asymmetrical bulges, extensive mismatches and 2'-O-methylation of ds-siRNA and miRNA interfered with processing. Whereas Mg(2+) was the cation that best supported the catalytic activity of RNase3, binding of 21 nt small dsRNA molecules was most efficient in the presence of Mn(2+). Processing of long dsRNA by RNase3 was efficient at pH 7.5 and 8.5, whereas ds-siRNA was processed more efficiently at pH 8.5. The results revealed factors that influence binding and processing of small dsRNA substrates by class 1 RNase III in vitro or make them unsuitable for processing by the enzyme.

Tyrosine phosphorylation of the triple gene block protein 3 regulates cell-to-cell movement and protein interactions of Potato mop-top virus.

Functions of viral proteins can be regulated through phosphorylation by serine/threonine kinases in plants, but little is known about the involvement of tyrosine kinases in plant virus infection. In this study, TGBp3, one of the three movement proteins encoded by a triple gene block (TGB) of Potato mop-top virus (PMTV), was detected for the first time in PMTV-infected plants and found to be tyrosine phosphorylated. Phosphorylation sites (Tyr(87-89) and Tyr(120)) were located in two amino acid motifs conserved in the TGB-containing, rod-shaped plant viruses. Substitution of these tyrosine residues in both motifs was needed to abolish tyrosine phosphorylation of TGBp3. Substitution of Tyr(87-89) with alanine residues enhanced the interaction between TGBp3 and TGBp2 and inhibited cell-to-cell movement of PMTV. On the other hand, substitution of Tyr(120) with alanine resulted in no alteration in the interaction of TGBp3 with TGBp2, but the mutant virus was not infectious. The results suggest that tyrosine phosphorylation is a mechanism regulating the functions of plant virus movement proteins.

fPoxDB: fungal peroxidase database for comparative genomics.

BACKGROUND: Peroxidases are a group of oxidoreductases which mediate electron transfer from hydrogen peroxide (H2O2) and organic peroxide to various electron acceptors. They possess a broad spectrum of impact on industry and fungal biology. There are numerous industrial applications using peroxidases, such as to catalyse highly reactive pollutants and to breakdown lignin for recycling of carbon sources. Moreover, genes encoding peroxidases play important roles in fungal pathogenicity in both humans and plants. For better understanding of fungal peroxidases at the genome-level, a novel genomics platform is required. To this end, Fungal Peroxidase Database (fPoxDB; http://peroxidase.riceblast.snu.ac.kr/) has been developed to provide such a genomics platform for this important gene family. DESCRIPTION: In order to identify and classify fungal peroxidases, 24 sequence profiles were built and applied on 331 genomes including 216 from fungi and Oomycetes. In addition, NoxR, which is known to regulate NADPH oxidases (NoxA and NoxB) in fungi, was also added to the pipeline. Collectively, 6,113 genes were predicted to encode 25 gene families, presenting well-separated distribution along the taxonomy. For instance, the genes encoding lignin peroxidase, manganese peroxidase, and versatile peroxidase were concentrated in the rot-causing basidiomycetes, reflecting their ligninolytic capability. As a genomics platform, fPoxDB provides diverse analysis resources, such as gene family predictions based on fungal sequence profiles, pre-computed results of eight bioinformatics programs, similarity search tools, a multiple sequence alignment tool, domain analysis functions, and taxonomic distribution summary, some of which are not available in the previously developed peroxidase resource. In addition, fPoxDB is interconnected with other family web systems, providing extended analysis opportunities. CONCLUSIONS: fPoxDB is a fungi-oriented genomics platform for peroxidases. The sequence-based prediction and diverse analysis toolkits with easy-to-follow web interface offer a useful workbench to study comparative and evolutionary genomics of peroxidases in fungi.

Functional diversification of duplicated chalcone synthase genes in anthocyanin biosynthesis of Gerbera hybrida.

• Chalcone synthase (CHS) is the key enzyme in the first committed step of the flavonoid biosynthetic pathway and catalyzes the stepwise condensation of 4-coumaroyl-CoA and malonyl-CoA to naringenin chalcone. In plants, CHS is often encoded by a small family of genes that are temporally and spatially regulated. Our earlier studies have shown that GCHS4 is highly activated by ectopic expression of an MYB-type regulator GMYB10 in gerbera (Gerbera hybrida). • The tissue- and development-specific expression patterns of three gerbera CHS genes were examined. Virus-induced gene silencing (VIGS) was used to knock down GCHS1 and GCHS4 separately in gerbera inflorescences. • Our data show that GCHS4 is the only CHS encoding gene that is expressed in the cyanidin-pigmented vegetative tissues of gerbera cv Terraregina. GCHS3 expression is pronounced in the pappus bristles of the flowers. Expression of both GCHS1 and GCHS4 is high in the epidermal cells of gerbera petals, but only GCHS1 is contributing to flavonoid biosynthesis. • Gerbera contains a family of three CHS encoding genes showing different spatial and temporal regulation. GCHS4 expression in gerbera petals is regulated post-transcriptionally, at the level of either translation elongation or protein stability.