The Pseudomonas syringae pv. tomato DC3000 effector HopD1 interferes with cellular dynamics associated with the function of the plant immune protein AtNHR2B.

The plant pathogenic bacterium Pseudomonas syringae pv tomato DC3000 (Pst DC3000) causes disease in tomato, in the model plant Arabidopsis thaliana, and conditionally in Nicotiana benthamiana. The pathogenicity of Pst DC3000 is mostly due to bacterial virulence proteins, known as effectors, that are translocated into the plant cytoplasm through the type III secretion system (T3SS). Bacterial type III secreted effectors (T3SEs) target plants physiological processes and suppress defense responses to enable and support bacterial proliferation. The Pst DC3000 T3SE HopD1 interferes with plant defense responses by targeting the transcription factor NTL9. This work shows that HopD1 also targets the immune protein AtNHR2B (Arabidopsis thaliana nonhost resistance 2B), a protein that localizes to dynamic vesicles of the plant endomembrane system. Live-cell imaging of Nicotiana benthamiana plants transiently co-expressing HopD1 fused to the epitope haemagglutinin (HopD1-HA) with AtNHR2B fused to the red fluorescent protein (AtNHR2B-RFP), revealed that HopD1-HA interferes with the abundance and cellular dynamics of AtNHR2B-RFP-containing vesicles. The results from this study shed light into an additional function of HopD1 while contributing to understanding how T3SEs also target vesicle trafficking-mediated processes in plants.

Agrobacterium expressing a type III secretion system delivers Pseudomonas effectors into plant cells to enhance transformation.

Agrobacterium-mediated plant transformation (AMT) is the basis of modern-day plant biotechnology. One major drawback of this technology is the recalcitrance of many plant species/varieties to Agrobacterium infection, most likely caused by elicitation of plant defense responses. Here, we develop a strategy to increase AMT by engineering Agrobacterium tumefaciens to express a type III secretion system (T3SS) from Pseudomonas syringae and individually deliver the P. syringae effectors AvrPto, AvrPtoB, or HopAO1 to suppress host defense responses. Using the engineered Agrobacterium, we demonstrate increase in AMT of wheat, alfalfa and switchgrass by ~250%-400%. We also show that engineered A. tumefaciens expressing a T3SS can deliver a plant protein, histone H2A-1, to enhance AMT. This strategy is of great significance to both basic research and agricultural biotechnology for transient and stable transformation of recalcitrant plant species/varieties and to deliver proteins into plant cells in a non-transgenic manner.

Functional role of formate dehydrogenase 1 (FDH1) for host and nonhost disease resistance against bacterial pathogens.

Nonhost disease resistance is the most common type of plant defense mechanism against potential pathogens. In the present study, the metabolic enzyme formate dehydrogenase 1 (FDH1) was identified to associate with nonhost disease resistance in Nicotiana benthamiana and Arabidopsis thaliana. In Arabidopsis, AtFDH1 was highly upregulated in response to both host and nonhost bacterial pathogens. The Atfdh1 mutants were compromised in nonhost resistance, basal resistance, and gene-for-gene resistance. The expression patterns of salicylic acid (SA) and jasmonic acid (JA) marker genes after pathogen infections in Atfdh1 mutant indicated that both SA and JA are involved in the FDH1-mediated plant defense response to both host and nonhost bacterial pathogens. Previous studies reported that FDH1 localizes to mitochondria, or both mitochondria and chloroplasts. Our results showed that the AtFDH1 mainly localized to mitochondria, and the expression level of FDH1 was drastically increased upon infection with host or nonhost pathogens. Furthermore, we identified the potential co-localization of mitochondria expressing FDH1 with chloroplasts after the infection with nonhost pathogens in Arabidopsis. This finding suggests the possible role of FDH1 in mitochondria and chloroplasts during defense responses against bacterial pathogens in plants.

The Pseudomonas syringae type III effector HopG1 triggers necrotic cell death that is attenuated by AtNHR2B.

The plant pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) has become a paradigm to investigate plant-bacteria interactions due to its ability to cause disease in the model plant Arabidopsis thaliana. Pst DC3000 uses the type III secretion system to deliver type III secreted effectors (T3SEs) directly into the plant cytoplasm. Pst DC3000 T3SEs contribute to pathogenicity by suppressing plant defense responses and targeting plant's physiological processes. Although the complete repertoire of effectors encoded in the Pst DC3000 genome have been identified, the specific function for most of them remains to be elucidated. Among those effectors, the mitochondrial-localized T3E HopG1, suppresses plant defense responses and promotes the development of disease symptoms. Here, we show that HopG1 triggers necrotic cell death that enables the growth of adapted and non-adapted pathogens. We further showed that HopG1 interacts with the plant immunity-related protein AtNHR2B and that AtNHR2B attenuates HopG1- virulence functions. These results highlight the importance of HopG1 as a multi-faceted protein and uncover its interplay with AtNHR2B.

Bacterial Panicle Blight and Burkholderia glumae: From Pathogen Biology to Disease Control.

Bacterial panicle blight (BPB), caused by the bacterium Burkholderia glumae, has affected rice production worldwide. Despite its importance, neither the disease nor the causal agent are well understood. Moreover, methods to manage BPB are still lacking. Nevertheless, the emerging importance of this pathogen has stimulated research to identify the mechanisms of pathogenicity, to gain insight into plant disease resistance, and to develop strategies to manage the disease. In this review, we consolidate current information regarding the virulence factors that have been identified in B. glumae and present a model of the disease and the pathogen. We also provide an update on the current research status to develop methods to control the disease especially through biological control approaches and through the development of resistant cultivars.

Harnessing Pseudomonas protegens to Control Bacterial Panicle Blight of Rice.

Bacterial panicle blight of rice is a seedborne disease caused by the bacterium Burkholderia glumae. This disease has affected rice production worldwide and its effects are likely to become more devastating with the continuous increase in global temperatures, especially during the growing season. The bacterium can cause disease symptoms in different tissues and at different developmental stages. In reproductive stages, the bacterium interferes with grain development in the panicles and, as a result, directly affects rice yield. Currently, there are no methods to control the disease because chemical control is not effective and completely resistant cultivars are not available. Thus, a promising approach is the use of antagonistic microorganisms. In this work, we identified one strain of Pseudomonas protegens and one strain of B. cepacia with antimicrobial activity against B. glumae in vitro and in planta. We further characterized the antimicrobial activity of P. protegens and found that this activity is associated with bacterial secretions. Cell-free secretions from P. protegens inhibited the growth of B. glumae in vitro and also prevented B. glumae from causing disease in rice. Although the specific molecules associated with these activities have not been identified, these findings suggest that the secreted fractions from P. protegens could be harnessed as biopesticides to control bacterial panicle blight of rice.

The Arabidopsis Proteins AtNHR2A and AtNHR2B Are Multi-Functional Proteins Integrating Plant Immunity With Other Biological Processes.

AtNHR2A (Arabidopsis thaliana nonhost resistance 2A) and AtNHR2B (Arabidopsis thaliana nonhost resistance 2B) are two proteins that participate in nonhost resistance, a broad-spectrum mechanism of plant immunity that protects plants against the majority of potential pathogens. AtNHR2A and AtNHR2B are localized to the cytoplasm, chloroplasts, and other subcellular compartments of unknown identity. The multiple localizations of AtNHR2A and AtNHR2B suggest that these two proteins are highly dynamic and versatile, likely participating in multiple biological processes. In spite of their importance, the specific functions of AtNHR2A and AtNHR2B have not been elucidated. Thus, to aid in the functional characterization of these two proteins and identify the biological processes in which these proteins operate, we used immunoprecipitation coupled with mass spectrometry (IP-MS) to identify proteins interacting with AtNHR2A and AtNHR2B and to generate their interactome network. Further validation of three of the identified proteins provided new insights into specific pathways and processes related to plant immunity where AtNHR2A and AtNHR2B participate. Moreover, the comprehensive analysis of the AtNHR2A- and AtNHR2B-interacting proteins using published empirical information revealed that the functions of AtNHR2A and AtNHR2B are not limited to plant immunity but encompass other biological processes.

Dissecting the functional domains of the Arabidopsis thaliana nonhost resistance 2B (AtNHR2B) protein.

The Arabidopsis thaliana nonhost resistant 2B (AtNHR2B) is involved in plant defense responses by mediating the deposition of the ß-1,3-glucan polymer callose to the cell wall in response to bacterial pathogens. Despite having a critical role in plant immunity, the exact mechanism of how this protein functions is not known and its protein sequence does not have any similarity to known proteins characterized to date. Using in silico analysis we identified three transmembrane domains and two nuclear localization signals (NLS). To validate these predictions, we generated truncated versions of the protein fused to the green fluorescent protein (GFP) to analyze their subcellular localization by laser scanning confocal microscopy. We found that the in silico predictions matched the subcellular localization of the truncated versions. Specifically, the presence of at least one of the transmembrane domain was required for membrane-bound subcellular compartments. Intriguingly, the localization of the transmembrane domains and the nuclear localization signals correspond to overlapping regions of the protein at the C-terminus and found one truncation that enabled protein localization to the nucleus. These results highlight that AtNHR2B is a unique protein composed of various domains that enable the protein to localize to diverse subcellular compartments and, by virtue of these multiple localizations, likely functions in multiple biological processes.

Nucleolar GTP-Binding Protein 1-2 (NOG1-2) Interacts with Jasmonate-ZIMDomain Protein 9 (JAZ9) to Regulate Stomatal Aperture during Plant Immunity.

Plant defense responses at stomata and apoplast are the most important early events during plant⁻bacteria interactions. The key components of stomatal defense responses have not been fully characterized. A GTPase encoding gene, NOG1-2, which is required for stomatal innate immunity against bacterial pathogens, was recently identified. Functional studies in Arabidopsis revealed that NOG1-2 regulates guard cell signaling in response to biotic and abiotic stimulus through jasmonic acid (JA)- and abscisic acid (ABA)-mediated pathways. Interestingly, in this study, Jasmonate-ZIM-domain protein 9 (JAZ9) was identified to interact with NOG1-2 for the regulation of stomatal closure. Upon interaction, JAZ9 reduces GTPase activity of NOG1-2. We explored the role of NOG1-2 binding with JAZ9 for COI1-mediated JA signaling and hypothesized that its function may be closely linked to MYC2 transcription factor in the regulation of the JA-signaling cascade in stomatal defense against bacterial pathogens. Our study provides valuable information on the function of a small GTPase, NOG1-2, in guard cell signaling and early plant defense in response to bacterial pathogens.

Two Chloroplast-Localized Proteins: AtNHR2A and AtNHR2B, Contribute to Callose Deposition During Nonhost Disease Resistance in Arabidopsis.

Plants are naturally resistant to most pathogens through a broad and durable defense response called nonhost disease resistance. Nonhost disease resistance is a complex process that includes preformed physical and chemical barriers and induced responses. In spite of its importance, many components of nonhost disease resistance remain to be identified and characterized. Using virus-induced gene silencing in Nicotiana benthamiana, we discovered a novel gene that we named NbNHR2 (N. benthamiana nonhost resistance 2). NbNHR2-silenced plants were susceptible to the nonadapted pathogen Pseudomonas syringae pv. tomato T1, which does not cause disease in wild-type or nonsilenced N. benthamiana plants. We found two orthologous genes in Arabidopsis thaliana: AtNHR2A and AtNHR2B. Similar to the results obtained in N. benthamiana, Atnhr2a and Atnhr2b mutants were susceptible to the nonadapted bacterial pathogen of A. thaliana, P. syringae pv. tabaci. We further found that these mutants were also defective in callose deposition. AtNHR2A and AtNHR2B fluorescent protein fusions transiently expressed in N. benthamiana localized predominantly to chloroplasts and a few unidentified dynamic puncta. RFP-AtNHR2A and AtNHR2B-GFP displayed overlapping signals in chloroplasts, indicating that the two proteins could interact, an idea supported by coimmunoprecipitation studies. We propose that AtNHR2A and AtNHR2B are new components of a chloroplast-signaling pathway that activates callose deposition to the cell wall in response to bacterial pathogens.

GENERAL CONTROL NONREPRESSIBLE4 Degrades 14-3-3 and the RIN4 Complex to Regulate Stomatal Aperture with Implications on Nonhost Disease Resistance and Drought Tolerance.

Plants have complex and adaptive innate immune responses against pathogen infections. Stomata are key entry points for many plant pathogens. Both pathogens and plants regulate stomatal aperture for pathogen entry and defense, respectively. Not all plant proteins involved in stomatal aperture regulation have been identified. Here, we report GENERAL CONTROL NONREPRESSIBLE4 (GCN4), an AAA+-ATPase family protein, as one of the key proteins regulating stomatal aperture during biotic and abiotic stress. Silencing of GCN4 in Nicotiana benthamiana and Arabidopsis thaliana compromises host and nonhost disease resistance due to open stomata during pathogen infection. AtGCN4 overexpression plants have reduced H+-ATPase activity, stomata that are less responsive to pathogen virulence factors such as coronatine (phytotoxin produced by the bacterium Pseudomonas syringae) or fusicoccin (a fungal toxin produced by the fungus Fusicoccum amygdali), reduced pathogen entry, and enhanced drought tolerance. This study also demonstrates that AtGCN4 interacts with RIN4 and 14-3-3 proteins and suggests that GCN4 degrades RIN4 and 14-3-3 proteins via a proteasome-mediated pathway and thereby reduces the activity of the plasma membrane H+-ATPase complex, thus reducing proton pump activity to close stomata.

The small GTPase, nucleolar GTP-binding protein 1 (NOG1), has a novel role in plant innate immunity.

Plant defense responses at stomata and apoplast are the most important early events during plant-bacteria interactions. The key components for the signaling of stomatal defense and nonhost resistance have not been fully characterized. Here we report the newly identified small GTPase, Nucleolar GTP-binding protein 1 (NOG1), functions for plant immunity against bacterial pathogens. Virus-induced gene silencing of NOG1 compromised nonhost resistance in N. benthamiana and tomato. Comparative genomic analysis showed that two NOG1 copies are present in all known plant species: NOG1-1 and NOG1-2. Gene downregulation and overexpression studies of NOG1-1 and NOG1-2 in Arabidopsis revealed the novel function of these genes in nonhost resistance and stomatal defense against bacterial pathogens, respectively. Specially, NOG1-2 regulates guard cell signaling in response to biotic and abiotic stimuli through jasmonic acid (JA)- and abscisic acid (ABA)-mediated pathways. The results here provide valuable information on the new functional role of small GTPase, NOG1, in guard cell signaling and early plant defense in response to bacterial pathogens.

Regulation of primary plant metabolism during plant-pathogen interactions and its contribution to plant defense.

Plants are constantly exposed to microorganisms in the environment and, as a result, have evolved intricate mechanisms to recognize and defend themselves against potential pathogens. One of these responses is the downregulation of photosynthesis and other processes associated with primary metabolism that are essential for plant growth. It has been suggested that the energy saved by downregulation of primary metabolism is diverted and used for defense responses. However, several studies have shown that upregulation of primary metabolism also occurs during plant-pathogen interactions. We propose that upregulation of primary metabolism modulates signal transduction cascades that lead to plant defense responses. In support of this thought, we here compile evidence from the literature to show that upon exposure to pathogens or elicitors, plants induce several genes associated with primary metabolic pathways, such as those involved in the synthesis or degradation of carbohydrates, amino acids and lipids. In addition, genetic studies have confirmed the involvement of these metabolic pathways in plant defense responses. This review provides a new perspective highlighting the relevance of primary metabolism in regulating plant defense against pathogens with the hope to stimulate further research in this area.

Arabidopsis heterotrimeric G-proteins play a critical role in host and nonhost resistance against Pseudomonas syringae pathogens.

Heterotrimeric G-proteins have been proposed to be involved in many aspects of plant disease resistance but their precise role in mediating nonhost disease resistance is not well understood. We evaluated the roles of specific subunits of heterotrimeric G-proteins using knock-out mutants of Arabidopsis Gα, Gß and Gγ subunits in response to host and nonhost Pseudomonas pathogens. Plants lacking functional Gα, Gß and Gγ1Gγ2 proteins displayed enhanced bacterial growth and disease susceptibility in response to host and nonhost pathogens. Mutations of single Gγ subunits Gγ1, Gγ2 and Gγ3 did not alter bacterial disease resistance. Some specificity of subunit usage was observed when comparing host pathogen versus nonhost pathogen. Overexpression of both Gα and Gß led to reduced bacterial multiplication of nonhost pathogen P. syringae pv. tabaci whereas overexpression of Gß, but not of Gα, resulted in reduced bacterial growth of host pathogen P. syringae pv. maculicola, compared to wild-type Col-0. Moreover, the regulation of stomatal aperture by bacterial pathogens was altered in Gα and Gß mutants but not in any of the single or double Gγ mutants. Taken together, these data substantiate the critical role of heterotrimeric G-proteins in plant innate immunity and stomatal modulation in response to P. syringae.

Several components of SKP1/Cullin/F-box E3 ubiquitin ligase complex and associated factors play a role in Agrobacterium-mediated plant transformation.

• Successful genetic transformation of plants by Agrobacterium tumefaciens requires the import of bacterial T-DNA and virulence proteins into the plant cell that eventually form a complex (T-complex). The essential components of the T-complex include the single stranded T-DNA, bacterial virulence proteins (VirD2, VirE2, VirE3 and VirF) and associated host proteins that facilitate the transfer and integration of T-DNA. The removal of the proteins from the T-complex is likely achieved by targeted proteolysis mediated by VirF and the plant ubiquitin proteasome complex. • We evaluated the involvement of the host SKP1/culin/F-box (SCF)-E3 ligase complex and its role in plant transformation. Gene silencing, mutant screening and gene expression studies suggested that the Arabidopsis homologs of yeast SKP1 (suppressor of kinetochore protein 1) protein, ASK1 and ASK2, are required for Agrobacterium-mediated plant transformation. • We identified the role for SGT1b (suppressor of the G2 allele of SKP1), an accessory protein that associates with SCF-complex, in plant transformation. We also report the differential expression of many genes that encode F-box motif containing SKP1-interacting proteins (SKIP) upon Agrobacterium infection. • We speculate that these SKIP genes could encode the plant specific F-box proteins that target the T-complex associated proteins for polyubiquitination and subsequent degradation by the 26S proteasome.

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.

STENOFOLIA regulates blade outgrowth and leaf vascular patterning in Medicago truncatula and Nicotiana sylvestris.

Dicot leaf primordia initiate at the flanks of the shoot apical meristem and extend laterally by cell division and cell expansion to form the flat lamina, but the molecular mechanism of lamina outgrowth remains unclear. Here, we report the identification of STENOFOLIA (STF), a WUSCHEL-like homeobox transcriptional regulator, in Medicago truncatula, which is required for blade outgrowth and leaf vascular patterning. STF belongs to the MAEWEST clade and its inactivation by the transposable element of Nicotiana tabacum cell type1 (Tnt1) retrotransposon insertion leads to abortion of blade expansion in the mediolateral axis and disruption of vein patterning. We also show that the classical lam1 mutant of Nicotiana sylvestris, which is blocked in lamina formation and stem elongation, is caused by deletion of the STF ortholog. STF is expressed at the adaxial-abaxial boundary layer of leaf primordia and governs organization and outgrowth of lamina, conferring morphogenetic competence. STF does not affect formation of lateral leaflets but is critical to their ability to generate a leaf blade. Our data suggest that STF functions by modulating phytohormone homeostasis and crosstalk directly linked to sugar metabolism, highlighting the importance of coordinating metabolic and developmental signals for leaf elaboration.

Agroinoculation and agroinfiltration: simple tools for complex gene function analyses.

Agroinoculation, first developed as a simple tool to study plant-virus interactions, is a popular method of choice for functional gene analysis of viral genomes. With the explosive growth of genomic information and the development of advanced vectors to dissect plant gene function, this reliable method of viral gene delivery in plants, has been recruited and morphed into a technique popularly known as agroinfiltration. This technique was developed to examine the effects of transient gene expression, with applications ranging from studies of plant-pathogen interactions, abiotic stresses, a variety of transient expression assays to study protein localization, and protein-protein interactions. We present a brief overview of literature which document both these applications, and then provide simple agroinoculation and agroinfiltration methods being used in our laboratory for functional gene analysis, as well as for fast-forward and reverse genetic screens using virus-induced gene silencing (VIGS).

Harpin mediates cell aggregation in Erwinia chrysanthemi 3937.

The hypersensitive response elicitor harpin (HrpN) of soft rot pathogen Erwinia chrysanthemi strains 3937 and EC16 is secreted via the type III secretion system and remains cell surface bound. Strain 3937 HrpN is essential for cell aggregation, but the C-terminal one-third of the protein is not required for aggregative activity.

The Erwinia chrysanthemi EC16 hrp/hrc gene cluster encodes an active Hrp type III secretion system that is flanked by virulence genes functionally unrelated to the Hrp system.

Erwinia chrysanthemi is a host-promiscuous plant pathogen that possesses a type III secretion system (TTSS) similar to that of the host-specific pathogens E. amylovora and Pseudomonas syringae. The regions flanking the TTSS-encoding hrp/hrc gene clusters in the latter pathogens encode various TTSS-secreted proteins. DNA sequencing of the complete E. chrysanthemi hrp/hrc gene cluster and approximately 12 kb of the flanking regions (beyond the previously characterized hecA adhesin gene in the left flank) revealed that the E. chrysanthemi TTSS genes were syntenic and similar (>50% amino-acid identity) with their E. amylovora orthologs. However, the hrp/hrc cluster was interrupted by a cluster of four genes, only one of which, a homolog of lytic transglycosylases, is implicated in TTSS functions. Furthermore, the regions flanking the hrp/hrc cluster lacked genes that were likely to encode TTSS substrates. Instead, some of the genes in these regions predict ABC transporters and methyl-accepting chemotaxis proteins that could have alternative roles in virulence. Mutations affecting all of the genes in the regions flanking or interrupting the hrp/hrc cluster were constructed in E. chrysanthemi CUCPB5047, a mutant whose reduced pectolytic capacity can enhance the phenotype of minor virulence factors. Mutants were screened in witloof chicory leaves and then in potato tubers and Nicotiana clevelandii seedlings. Mu dII1734 insertion in one gene, designated virA, resulted in strongly reduced virulence in all three tests. virA is immediately downstream of hecA, has an unusually low G+C content of 38%, and predicts an unknown protein of 111 amino acids. The E. chrysanthemi TTSS was shown to be active by its ability to translocate AvrPto-Cya (a P. syringae TTSS effector fused to an adenylate cyclase reporter that is active in the presence of eukaryote calmodulin) into N. benthamiana leaf cells. However, VirA(1-61)-Cya was not translocated into plant cells, and virA expression was not affected by mutations in E. chrysanthemi Hrp regulator genes hrpL and hrpS. Thus, the 44-kb region of the E. chrysanthemi EC16 genome that is centered on the hrplhrc cluster encodes a potpourri of virulence factors, but none of these appear to be a TTSS effector.