Virus-induced gene silencing database for phenomics and functional genomics in Nicotiana benthamiana.

Virus-induced gene silencing (VIGS) is an important forward and reverse genetics method for the study of gene function in many plant species, especially Nicotiana benthamiana. However, despite the widespread use of VIGS, a searchable database compiling the phenotypes observed with this method is lacking. Such a database would allow researchers to know the phenotype associated with the silencing of a large number of individual genes without experimentation. We have developed a VIGS phenomics and functional genomics database (VPGD) that has DNA sequence information derived from over 4,000 N. benthamiana VIGS clones along with the associated silencing phenotype for approximately 1,300 genes. The VPGD has a built-in BLAST search feature that provides silencing phenotype information of specific genes. In addition, a keyword-based search function could be used to find a specific phenotype of interest with the corresponding gene, including its Gene Ontology descriptions. Query gene sequences from other plant species that have not been used for VIGS can also be searched for their homologs and silencing phenotype in N. benthamiana. VPGD is useful for identifying gene function not only in N. benthamiana but also in related Solanaceae plants such as tomato and potato. The database is accessible at http://vigs.noble.org.

Plant Ribosomal Proteins, RPL12 and RPL19, Play a Role in Nonhost Disease Resistance against Bacterial Pathogens.

Characterizing the molecular mechanism involved in nonhost disease resistance is important to understand the adaptations of plant-pathogen interactions. In this study, virus-induced gene silencing (VIGS)-based forward genetics screen was utilized to identify genes involved in nonhost resistance in Nicotiana benthamiana. Genes encoding ribosomal proteins, RPL12 and RPL19, were identified in the screening. These genes when silenced in N. benthamiana caused a delay in nonhost bacteria induced hypersensitive response (HR) with concurrent increase in nonhost bacterial multiplication. Arabidopsis mutants of AtRPL12 and AtRPL19 also compromised nonhost resistance. The studies on NbRPL12 and NbRPL19 double silenced plants suggested that both RPL12 and RPL19 act in the same pathway to confer nonhost resistance. Our work suggests a role for RPL12 and RPL19 in nonhost disease resistance in N. benthamiana and Arabidopsis. In addition, we show that these genes also play a minor role in basal resistance against virulent pathogens.

AtCYP710A1 gene-mediated stigmasterol production plays a role in imparting temperature stress tolerance in Arabidopsis thaliana.

Stigmasterol and sitosterol, important sterols present in plants, are known to influence permeability and fluidity characteristics of the plasma membrane and other organellar membranes. We had previously demonstrated that the Arabidopsis Atcyp710A1 gene, which catalyzes conversion of sitosterol into stigmasterol, plays a role in plasma membrane permeability, thus influencing leakage of cellular nutrients and ions into apoplast. In this study, we investigated the role of this gene in imparting various abiotic stress tolerances in Arabidopsis. By analyzing Atcyp710a1 mutant and AtCYP710A1 overexpressor lines, we found that the AtCYP710A1 gene plays a role in imparting low and high temperature tolerance.

Phytosterols play a key role in plant innate immunity against bacterial pathogens by regulating nutrient efflux into the apoplast.

Bacterial pathogens colonize a host plant by growing between the cells by utilizing the nutrients present in apoplastic space. While successful pathogens manipulate the plant cell membrane to retrieve more nutrients from the cell, the counteracting plant defense mechanism against nonhost pathogens to restrict the nutrient efflux into the apoplast is not clear. To identify the genes involved in nonhost resistance against bacterial pathogens, we developed a virus-induced gene-silencing-based fast-forward genetics screen in Nicotiana benthamiana. Silencing of N. benthamiana SQUALENE SYNTHASE, a key gene in phytosterol biosynthesis, not only compromised nonhost resistance to few pathovars of Pseudomonas syringae and Xanthomonas campestris, but also enhanced the growth of the host pathogen P. syringae pv tabaci by increasing nutrient efflux into the apoplast. An Arabidopsis (Arabidopsis thaliana) sterol methyltransferase mutant (sterol methyltransferase2) involved in sterol biosynthesis also compromised plant innate immunity against bacterial pathogens. The Arabidopsis cytochrome P450 CYP710A1, which encodes C22-sterol desaturase that converts ß-sitosterol to stigmasterol, was dramatically induced upon inoculation with nonhost pathogens. An Arabidopsis Atcyp710A1 null mutant compromised both nonhost and basal resistance while overexpressors of AtCYP710A1 enhanced resistance to host pathogens. Our data implicate the involvement of sterols in plant innate immunity against bacterial infections by regulating nutrient efflux into the apoplast.

Glycolate oxidase modulates reactive oxygen species-mediated signal transduction during nonhost resistance in Nicotiana benthamiana and Arabidopsis.

In contrast to gene-for-gene disease resistance, nonhost resistance governs defense responses to a broad range of potential pathogen species. To identify specific genes involved in the signal transduction cascade associated with nonhost disease resistance, we used a virus-induced gene-silencing screen in Nicotiana benthamiana, and identified the peroxisomal enzyme glycolate oxidase (GOX) as an essential component of nonhost resistance. GOX-silenced N. benthamiana and Arabidopsis thaliana GOX T-DNA insertion mutants are compromised for nonhost resistance. Moreover, Arabidopsis gox mutants have lower H(2)O(2) accumulation, reduced callose deposition, and reduced electrolyte leakage upon inoculation with hypersensitive response-causing nonhost pathogens. Arabidopsis gox mutants were not affected in NADPH oxidase activity, and silencing of a gene encoding NADPH oxidase (Respiratory burst oxidase homolog) in the gox mutants did not further increase susceptibility to nonhost pathogens, suggesting that GOX functions independently from NADPH oxidase. In the two gox mutants examined (haox2 and gox3), the expression of several defense-related genes upon nonhost pathogen inoculation was decreased compared with wild-type plants. Here we show that GOX is an alternative source for the production of H(2)O(2) during both gene-for-gene and nonhost resistance responses.

SGT1 contributes to coronatine signaling and Pseudomonas syringae pv. tomato disease symptom development in tomato and Arabidopsis.

• Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) causes an economically important bacterial speck disease on tomato and produces symptoms with necrotic lesions surrounded by chlorosis. The chlorosis is mainly attributed to a jasmonic acid (JA)-isoleucine analogue, coronatine (COR), produced by Pst DC3000. However, the molecular processes underlying lesion development and COR-induced chlorosis are poorly understood. • In this study, we took advantage of a chlorotic phenotype elicited by COR on Nicotiana benthamiana leaves and virus-induced gene silencing (VIGS) as a rapid reverse genetic screening tool and identified a role for SGT1 (suppressor of G2 allele of skp1) in COR-induced chlorosis. • Silencing of SGT1 in tomato resulted in reduction of disease-associated symptoms (cell death and chlorosis), suggesting a molecular connection between COR-induced chlorosis and cell death. In Arabidopsis, AtSGT1b but not AtSGT1a was required for COR responses, including root growth inhibition and Pst DC3000 symptom (water soaked lesion) development. Notably, overexpression of AtSGT1b did not alter Pst DC3000 symptoms or sensitivity to COR. • Taken together, our results demonstrate that SGT1/SGT1b is required for COR-induced chlorosis and subsequent necrotic disease development in tomato and Arabidopsis. SGT1 is therefore a component of the COR/JA-mediated signal transduction pathway.

SGT1 positively regulates the process of plant cell death during both compatible and incompatible plant-pathogen interactions.

SGT1 (suppressor of G2 allele of Skp1), an interactor of SCF (Skp1-Cullin-F-box) ubiquitin ligase complexes that mediate protein degradation, plays an important role at both G1-S and G2-M cell cycle transitions in yeast, and is highly conserved throughout eukaryotes. Plant SGT1 is required for both resistance (R) gene-mediated disease resistance and nonhost resistance to certain pathogens. Using virus-induced gene silencing (VIGS) in Nicotiana benthamiana, we demonstrate that SGT1 positively regulates the process of cell death during both host and nonhost interactions with various pathovars of Pseudomonas syringae. Silencing of NbSGT1 in N. benthamiana plants delays the induction of hypersensitive response (HR)-mediated cell death against nonhost pathogens and the development of disease-associated cell death caused by the host pathogen P. syringae pv. tabaci. Our results further demonstrate that NbSGT1 is required for Erwinia carotovora- and Sclerotinia sclerotiorum-induced disease-associated cell death. Overexpression of NbSGT1 in N. benthamiana accelerates the development of HR during R gene-mediated disease resistance and nonhost resistance. Our data also indicate that SGT1 is required for pathogen-induced cell death, but is not always necessary for the restriction of bacterial multiplication in planta. Therefore, we conclude that SGT1 is an essential component affecting the process of cell death during both compatible and incompatible plant-pathogen interactions.

Overexpression of a fatty acid amide hydrolase compromises innate immunity in Arabidopsis.

N-acylethanolamines are a group of lipid mediators that accumulate under a variety of neurological and pathological conditions in mammals. N-acylethanolamine signaling is terminated by the action of diverse hydrolases, among which fatty acid amide hydrolase (FAAH) has been well characterized. Here, we show that transgenic Arabidopsis lines overexpressing an AtFAAH are more susceptible to the bacterial pathogens Pseudomonas syringae pv. tomato and P. syringae pv. maculicola. AtFAAH overexpressors also were highly susceptible to non-host pathogens P. syringae pv. syringae and P. syringae pv. tabaci. AtFAAH overexpressors had lower amounts of jasmonic acid, abscisic acid and both free and conjugated salicylic acid (SA), compared with the wild-type. Gene expression studies revealed that transcripts of a number of plant defense genes, as well as genes involved in SA biosynthesis and signaling, were lower in AtFAAH overexpressors than wild-type plants. Our data suggest that FAAH overexpression alters phytohormone accumulation and signaling which in turn compromises innate immunity to bacterial pathogens.

Monitoring in planta bacterial infection at both cellular and whole-plant levels using the green fluorescent protein variant GFPuv.

* Green fluorescent protein (GFP) labeling of bacteria has been used to study their infection of and localization in plants, but strong autofluorescence from leaves and the relatively weak green fluorescence of GFP-labeled bacteria restrict its broader application to investigations of plant-bacterial interactions. * A stable and broad-host-range plasmid vector (pDSK-GFPuv) that strongly expresses GFPuv protein was constructed not only for in vivo monitoring of bacterial infection, localization, activity, and movement at the cellular level under fluorescence microscopy, but also for monitoring bacterial disease development at the whole-plant level under long-wavelength ultraviolet (UV) light. * The presence of pDSK-GFPuv did not have significant impact on the in vitro or in planta growth and virulence of phytobacteria. A good correlation between bacterial cell number and fluorescence intensity was observed, which allowed us to rapidly estimate the bacterial population in plant leaf tissue. We demonstrated that GFPuv-expressing bacteria can be used to screen plants that are compromised for nonhost disease resistance and Agrobacterium attachment. * The use of GFPuv-labeled bacteria has a wide range of applications in host-bacterial interaction studies and bacterial ecology-related research.

Identification and characterization of plant genes involved in Agrobacterium-mediated plant transformation by virus-induced gene silencing.

Genetic transformation of plant cells by Agrobacterium tumefaciens represents a unique case of trans-kingdom sex requiring the involvement of both bacterial virulence proteins and plant-encoded proteins. We have developed in planta and leaf-disk assays in Nicotiana benthamiana for identifying plant genes involved in Agrobacterium-mediated plant transformation using virus-induced gene silencing (VIGS) as a genomics tool. VIGS was used to validate the role of several genes that are either known or speculated to be involved in Agrobacterium-mediated plant transformation. We showed the involvement of a nodulin-like protein and an alpha-expansin protein (alpha-Exp) during Agrobacterium infection. Our data suggest that alpha-Exp is involved during early events of Agrobacterium-mediated transformation but not required for attaching A. tumefaciens. By employing the combination of the VIGS-mediated forward genetics approach and an in planta tumorigenesis assay, we identified 21 ACG (altered crown gall) genes that, when silenced, produced altered crown gall phenotypes upon infection with a tumorigenic strain of A. tumefaciens. One of the plant genes identified from the screening, Histone H3 (H3), was further characterized for its biological role in Agrobacterium-mediated plant transformation. We provide evidence for the role of H3 in transfer DNA integration. The data presented here suggest that the VIGS-based approach to identify and characterize plant genes involved in genetic transformation of plant cells by A. tumefaciens is simple, rapid, and robust and complements other currently used approaches.

Involvement of quinolinate phosphoribosyl transferase in promotion of potato growth by a Burkholderia strain.

Burkholderia sp. strain PsJN stimulates root growth of potato explants compared to uninoculated controls under gnotobiotic conditions. In order to determine the mechanism by which this growth stimulation occurs, we used Tn5 mutagenesis to produce a mutant, H41, which exhibited no growth-promoting activity but was able to colonize potato plants as well as the wild-type strain. The gene associated with the loss of growth promotion in H41 was shown to exhibit 65% identity at the amino acid level to the nadC gene encoding quinolinate phosphoribosyltransferase (QAPRTase) in Ralstonia solanacearum. Complementation of H41 with QAPRTase restored growth promotion of potato explants by this mutant. Expression of the gene identified in Escherichia coli yielded a protein with QAPRTase activities that catalyzed the de novo formation of nicotinic acid mononucleotide (NaMN). Two other genes involved in the same enzymatic pathway, nadA and nadB, were physically linked to nadC. The nadA gene was cotranscribed with nadC as an operon in wild-type strain PsJN, while the nadB gene was located downstream of the nadA-nadC operon. Growth promotion by H41 was fully restored by addition of NaMN to the tissue culture medium. These data suggested that QAPRTase may play a role in the signal pathway for promotion of plant growth by PsJN.

Clover proliferation phytoplasma: 'Candidatus Phytoplasma trifolii'.

Clover proliferation phytoplasma (CPR) is designated as the reference strain for the CP phylogenetic group or subclade, on the basis of molecular analyses of genomic DNA, the 16S rRNA gene and the 16S-23S spacer region. Other strains related to CPR include alfalfa witches'-broom (AWB), brinjal little leaf (BLL), beet leafhopper-transmitted virescence (BLTV), Illinois elm yellows (ILEY), potato witches'-broom (PWB), potato yellows (PY), tomato big bud in California (TBBc) and phytoplasmas from Fragaria multicipita (FM). Phylogenetic analysis of the 16S rRNA gene sequences of BLL, CPR, FM and ILEY, together with sequences from 16 other phytoplasmas that belong to the ash yellows (AshY), jujube witches'-broom (JWB) and elm yellows (EY) groups that were available in GenBank, produced a tree on which these phytoplasmas clearly clustered as a discrete group. Three subgroups have been classified on the basis of sequence homology and the collective RFLP patterns of amplified 16S rRNA genes. AWB, BLTV, PWB and TBBc are assigned to taxonomic subgroup CP-A, FM belongs to subgroup CP-B and BLL and ILEY are assigned to subgroup CP-C. Genetic heterogeneity between different isolates of AWB, CPR and PWB has been observed from heteroduplex mobility assay analysis of amplified 16S rRNA genes and the 16S-23S spacer region. Two unique signature sequences that can be utilized to distinguish the CP group from others were present. On the basis of unique properties of the DNA from clover proliferation phytoplasma, the name 'Candidatus Phytoplasma trifolii' is proposed for the CP group.

Molecular Characterization and Classification of Phytoplasmas Associated with Canola Yellows and a New Phytoplasma Strain Associated with Dandelions.

DNA isolated from symptomatic canola (Brassica napus, Brassica rapa) and dandelion (Taraxacum officinale) was used to amplify 16S ribosomal DNA fragments by polymerase chain reaction using two pairs of universal primers P1/P6 and R16F2n/R2. Restriction fragment length polymorphism (RFLP) analysis of the amplified DNA fragments using endonucleases AluI, HhaI, HpaII, MseI, RsaI, and Sau 3AI revealed two distinct types of phytoplasmas in canola with similar symptoms. One had the same RFLP profiles as the phytoplasmas in subgroup 16SrI-A, whereas the other one had RFLP profiles similar to those of phytoplasmas in subgroup 16SrI-B. Phytoplasmas were detected in symptomatic dandelion plants that were collected from canola and alfalfa fields where severe alfalfa witches'-broom occurred. Comparative studies indicated that two different phytoplasmas were associated with the dandelion plants. One was identified as a member of subgroup 16SrI-A, whereas another one was classified as a member of a distinct subgroup in the aster yellows group on the basis of the unique RFLP patterns.