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.

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 β-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.

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.

Summary• 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 GFPuvexpressing 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 hostbacterial interaction studies and bacterial ecology-related research.

SummaryN-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.

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.

Clover proliferation phytoplasma (CP R ) 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 CP R 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, CP R , 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, CP R 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.

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.