Jacek Hennig

Temperature-Dependent Induction of Salicylic Acid and Its Conjugates during the Resistance Response to Tobacco Mosaic Virus Infection.

Increases in endogenous salicylic acid (SA) levels and induction of several families of pathogenesis-related genes (PR-1 through PR-5) occur during the resistance response of tobacco to tobacco mosaic virus infection. We found that at temperatures that prevent the induction of PR genes and resistance, the increases in SA levels were eliminated. The addition of exogenous SA to infected plants at these temperatures was sufficient to induce the PR genes but not the hypersensitive response. However, when the resistance response was restored by shifting infected plants to permissive temperatures, SA levels increased dramatically and preceded PR-1 gene expression and necrotic lesion formation associated with resistance. SA was also found in a conjugated form whose levels increased in parallel with the free SA levels. The majority of the conjugates appeared to be SA glucosides. The same glucoside was formed when plants were supplied with exogenous SA. These results provide further evidence that endogenous SA signals the induction of certain defense responses and suggests additional complexity in the modulation of this signal.

The Role of Annexin 1 in Drought Stress in Arabidopsis

Annexins act as targets of calcium signals in eukaryotic cells, and recent results suggest that they play an important role in plant stress responses. We found that in Arabidopsis (Arabidopsis thaliana), AnnAt1 (for annexin 1) mRNA levels were up-regulated in leaves by most of the stress treatments applied. Plants overexpressing AnnAt1 protein were more drought tolerant and knockout plants were more drought sensitive than ecotype Columbia plants. We also observed that hydrogen peroxide accumulation in guard cells was reduced in overexpressing plants and increased in knockout plants both before and after treatment with abscisic acid. Oxidative protection resulting from AnnAt1 overexpression could be due to the low level of intrinsic peroxidase activity exhibited by this protein in vitro, previously linked to a conserved histidine residue found in a peroxidase-like motif. However, analyses of a mutant H40A AnnAt1 protein in a bacterial complementation test and in peroxidase activity assays indicate that this residue is not critical to the ability of AnnAt1 to confer oxidative protection. To further examine the mechanism(s) linking AnnAt1 expression to stress resistance, we analyzed the reactive S3 cluster to determine if it plays a role in AnnAt1 oligomerization and/or is the site for posttranslational modification. We found that the two cysteine residues in this cluster do not form intramolecular or intermolecular bonds but are highly susceptible to oxidation-driven S-glutathionylation, which decreases the Ca2+ affinity of AnnAt1 in vitro. Moreover, S-glutathionylation of AnnAt1 occurs in planta after abscisic acid treatment, which suggests that this modification could be important in regulating the cellular function of AnnAt1 during stress responses.

Phosphorylation of HopQ1, a Type III Effector from Pseudomonas syringae, Creates a Binding Site for Host 14-3-3 Proteins

A pathogen effector protein undergoes specific phosphorylation and associates with host 14-3-3 proteins, which affects its subcellular distribution and stability. HopQ1 (for Hrp outer protein Q), a type III effector secreted by Pseudomonas syringae pv phaseolicola, is widely conserved among diverse genera of plant bacteria. It promotes the development of halo blight in common bean (Phaseolus vulgaris). However, when this same effector is injected into Nicotiana benthamiana cells, it is recognized by the immune system and prevents infection. Although the ability to synthesize HopQ1 determines host specificity, the role it plays inside plant cells remains unexplored. Following transient expression in planta, HopQ1 was shown to copurify with host 14-3-3 proteins. The physical interaction between HopQ1 and 14-3-3a was confirmed in planta using the fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique. Moreover, mass spectrometric analyses detected specific phosphorylation of the canonical 14-3-3 binding site (RSXpSXP, where pS denotes phosphoserine) located in the amino-terminal region of HopQ1. Amino acid substitution within this motif abrogated the association and led to altered subcellular localization of HopQ1. In addition, the mutated HopQ1 protein showed reduced stability in planta. These data suggest that the association between host 14-3-3 proteins and HopQ1 is important for modulating the properties of this bacterial effector.

Effect of Yeast CTA1 Gene Expression on Response of Tobacco Plants to Tobacco Mosaic Virus Infection

The response of tobacco (Nicotiana tabacum L. cv Xanthi-nc) plants with elevated catalase activity was studied after infection by tobacco mosaic virus (TMV). These plants contain the yeast (Saccharomyces cerevisiae) peroxisomal catalase geneCTA1 under the control of the cauliflower mosaic virus 35S promoter. The transgenic lines exhibited 2- to 4-fold higher total in vitro catalase activity than untransformed control plants under normal growth conditions. Cellular localization of the CTA1 protein was established using immunocytochemical analysis. Gold particles were detected mainly inside peroxisomes, whereas no significant labeling was detected in other cellular compartments or in the intercellular space. The physiological state of the transgenic plants was evaluated in respect to growth rate, general appearance, carbohydrate content, and dry weight. No significant differences were recorded in comparison with non-transgenic tobacco plants. The 3,3′-diaminobenzidine-stain method was applied to visualize hydrogen peroxide (H2O2) in the TMV infected tissue. Presence of H2O2 could be detected around necrotic lesions caused by TMV infection in non-transgenic plants but to a much lesser extent in the CTA1 transgenic plants. In addition, the size of necrotic lesions was significantly bigger in the infected leaves of the transgenic plants. Changes in the distribution of H2O2 and in lesion formation were not reflected by changes in salicylic acid production. In contrast to the local response, the systemic response in upper noninoculated leaves of both CTA1 transgenic and control plants was similar. This suggests that increased cellular catalase activity influences local but not systemic response to TMV infection.

Nucleocytoplasmic partitioning of tobacco N receptor is modulated by SGT1

SGT1 (Suppressor of G2 allele of SKP1) is required to maintain plant disease Resistance (R) proteins with Nucleotide-Binding (NB) and Leucine-Rich Repeat (LRR) domains in an inactive but signaling-competent state. SGT1 is an integral component of a multi-protein network that includes RACK1, Rac1, RAR1, Rboh, HSP90 and HSP70, and in rice the Mitogen-Activated Protein Kinase (MAPK), OsMAPK6. Tobacco (Nicotiana tabacum) N protein, which belongs to the Toll-Interleukin Receptor (TIR)-NB-LRR class of R proteins, confers resistance to Tobacco Mosaic Virus (TMV). Following transient expression in planta, we analyzed the functional relationship between SGT1, SIPK - a tobacco MAPK6 ortholog - and N, using mass spectrometry, confocal microscopy and pathogen assays. Here, we show that tobacco SGT1 undergoes specific phosphorylation in a canonical MAPK target-motif by SIPK. Mutation of this motif to mimic SIPK phosphorylation leads to an increased proportion of cells displaying SGT1 nuclear accumulation and impairs N-mediated resistance to TMV, as does phospho-null substitution at the same residue. Forced nuclear localization of SGT1 causes N to be confined to nuclei. Our data suggest that one mode of regulating nucleocytoplasmic partitioning of R proteins is by maintaining appropriate levels of SGT1 phosphorylation catalyzed by plant MAPK.

Is annexin 1 a multifunctional protein during stress responses

Accumulating evidence suggest that certain annexins can play a role in abiotic stress responses in plants. We found that for one member of the Arabidopsis annexin gene family, annexin 1 (AnnAt1), loss-of-function mutants are more sensitive to drought stress and gain-of-function mutants are more tolerant1. We also found that AnnAt1 is able to regulate accumulation of H2O2 in vivo in Arabidopsis cells based on the observation that the level of ROS accumulation following induction by ABA correlates with the level of AnnAt1 protein in transgenic Arabidopsis plants. Here we provide more commentary on the antioxidant activity of AnnAt1, critically assess the evidence that AnnAt1 and other annexins possess peroxidase activity, emphasize a redox-induced post-translational modification which occurs to AnnAt1 during ABA signaling, and discuss ways this annexin’s membrane associations could mediate stress signaling while addressing the potential that AnnAt1 is a multifunctional protein in plants.

Potato Annexin STANN1 Promotes Drought Tolerance and Mitigates Light Stress in Transgenic Solanum tuberosum L. Plants.

Annexins are a family of calcium- and membrane-binding proteins that are important for plant tolerance to adverse environmental conditions. Annexins function to counteract oxidative stress, maintain cell redox homeostasis, and enhance drought tolerance. In the present study, an endogenous annexin, STANN1, was overexpressed to determine whether crop yields could be improved in potato (Solanum tuberosum L.) during drought. Nine potential potato annexins were identified and their expression characterized in response to drought treatment. STANN1 mRNA was constitutively expressed at a high level and drought treatment strongly increased transcription levels. Therefore, STANN1 was selected for overexpression analysis. Under drought conditions, transgenic potato plants ectopically expressing STANN1 were more tolerant to water deficit in the root zone, preserved more water in green tissues, maintained chloroplast functions, and had higher accumulation of chlorophyll b and xanthophylls (especially zeaxanthin) than wild type (WT). Drought-induced reductions in the maximum efficiency and the electron transport rate of photosystem II (PSII), as well as the quantum yield of photosynthesis, were less pronounced in transgenic plants overexpressing STANN1 than in the WT. This conferred more efficient non-photochemical energy dissipation in the outer antennae of PSII and probably more efficient protection of reaction centers against photooxidative damage in transgenic plants under drought conditions. Consequently, these plants were able to maintain effective photosynthesis during drought, which resulted in greater productivity than WT plants despite water scarcity. Although the mechanisms underlying this stress protection are not yet clear, annexin-mediated photoprotection is probably linked to protection against light-induced oxidative stress.

Emerging role of SGT1 as a regulator of NB-LRR-receptor nucleocytoplasmic partitioning

Plant nucleotide-binding (NB) and leucine-rich repeat (LRR) receptors mediate effector-triggered immunity. Two major classes of NB-LRR proteins are involved in this process, namely, toll-interleukin receptor (TIR)-NB-LRR and coiled coil (CC)-NB-LRR proteins. Recent reports show that some of the TIR-NB-LRRs and CC-NB-LRRs localize to the cytoplasm and nucleus. Equilibrium between these pools is required for full resistance, suggesting tight regulation of nucleocytoplasmic receptor shuttling. We recently showed that SGT1, a protein that controls NB-LRR receptor stability and activity, facilitates nuclear import of N protein, which is a TIR-NB-LRR receptor. In this addendum, we show that the subcellular localization of Rx, a CC-NB-LRR protein, reflects the positions of SGT1 ectopic variants in the cell. This suggests that SGT1 might have a general role in maintaining the nucleocytoplasmic balance of NB-LRR receptors. We discuss these results in light of differences in the N and Rx systems of effector-triggered immunity.

The Salicylic Acid Signal for Activation of Plant Defenses is Mediated by Active Oxygen Species

A major objective of our research is to understand how plants perceive and then respond to pathogenic attack at the molecular and cellular levels. As a model system for our studies we have chosen the interaction of tobacco mosaic virus (TMV; one of the best characterized RNA viruses) with its host Nicotiana tabacum. Infection of tobacco plants with TMV results in one of two distinct responses which depends upon the genetic background of the host and the viral strain. In a susceptible tobacco cultivar (e.g. Xanthi) infection with TMV (e.g. strain U1) results in the systemic spread of virus from the original point of entry to distal parts of the host, with the potential for causing widespread damage. In contrast, tobacco cultivars (e.g. Xanthi nc) that carry a dominant resistance gene (e.g. N locus) restrict the spread of TMV to a small zone of tissue around the point of entry, where a necrotic lesion later appears. This resistance phenotype is called the “hypersensitive response” (HR) and is subsequently accompanied by the induction throughout the plant of systemic acquired resistance (SAR). Establishment of SAR results in enhanced resistance to a secondary challenge by the same (e.g. TMV) or even unrelated pathogens. Both the HR and SAR are correlated with the synthesis of abundant amounts of host-encoded, pathogenesis-related (PR) proteins. The production of these proteins seems to be part of a general defense system against pathogens, for their synthesis can also be induced by certain bacteria and fungi.

Induction, modification, and reception of the salicylic acid signal in plant defense.

Endogenous salicylic acid (SA) levels increase and several families of pathogenesis-related genes (including PR-1 and PR-2) are induced during the resistance response of tobacco to tobacco mosaic virus (TMV) infection. We have found that at a temperature (32 degrees C) that prevents the induction of PR genes and resistance, the increases in SA levels were eliminated. However, when the resistance response was restored by shifting inoculated plants to lower temperatures, SA levels increased dramatically and preceded PR-1 gene expression and necrotic lesion formation associated with resistance. SA was also found in a conjugated form whose levels increased in parallel with the free SA levels. This SA beta-glucoside (SAG) was as active as SA in inducing PR-1 gene expression. PR-1 gene induction by SAG was preceded by a transient release of SA. The existence of a mechanism that releases SA from SAG suggests a possible role for SAG in the maintenance of systemic acquired resistance. Previously, we identified a soluble salicylic acid-binding protein (SABP) in tobacco whose properties suggest that it may play a role in transmitting the SA signal during plant defence responses. This SABP has been purified 250-fold by sequential chromatography on DEAE-Sephacel, Sephacryl S-300, Blue Dextran-Agarose and Superose 6. Several monoclonal antibodies (mAbs) raised against the highly purified SABP immunoprecipitated the SA-binding activity and a 280 kDa protein. This 280 kDa protein also co-purified with the SA-binding activity during the various chromatography steps, suggesting that it was responsible for binding SA. Immunoblot analysis with the SABP-specific mAbs also detected the 280 kDa protein in highly purified preparations of SABP. However, in crude homogenates these mAbs only recognized a 57 kDa protein. These and other results suggest that SABP is a multimeric complex which contains, at least, a 57 kDa protein and whose components are readily cross-linked during purification.