Differential effectiveness of salicylate-dependent and jasmonate/ethylene-dependent induced resistance in Arabidopsis

Salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are each involved in the regulation of basal resistance against different pathogens. These three signals play important roles in induced resistance as well. SA is a key regulator of pathogen-induced systemic acquired resistance (SAR), whereas JA and ET are required for rhizobacteria-mediated induced systemic resistance (ISR). Both types of induced resistance are effective against a broad spectrum of pathogens. In this study, we compared the spectrum of effectiveness of SAR and ISR using an oomycete, a fungal, a bacterial, and a viral pathogen. In noninduced Arabidopsis plants, these pathogens are primarily resisted through either SA-dependent basal resistance (Peronospora parasitica and Turnip crinkle virus [TCV]), JA/ET-dependent basal resistance responses (Alternaria brassicicola), or a combination of SA-, JA-, and ET-dependent defenses (Xanthomonas campestris pv. armoraciae). Activation of ISR resulted in a significant level of protection against A. brassicicola, whereas SAR was ineffective against this pathogen. Conversely, activation of SAR resulted in a high level of protection against P. parasitica and TCV, whereas ISR conferred only weak and no protection against P. parasitica and TCV, respectively. Induction of SAR and ISR was equally effective against X. campestris pv. armoraciae. These results indicate that SAR is effective against pathogens that in noninduced plants are resisted through SA-dependent defenses, whereas ISR is effective against pathogens that in noninduced plants are resisted through JA/ET-dependent defenses. This suggests that SAR and ISR constitute a reinforcement of extant SA- or JA/ET-dependent basal defense responses, respectively.     

Silencing of the mitogen-activated protein kinase MPK6 compromises disease resistance in Arabidopsis

Here, we use a loss-of-function approach to demonstrate that the Arabidopsis (Arabidopsis thaliana) mitogen-activated protein kinase (MAPK) MPK6 plays a role in resistance to certain pathogens. MPK6-silenced Arabidopsis showed no apparent morphological phenotype or reduced fertility, indicating MPK6 is not required for development. However, resistances to an avirulent strain of Peronospora parasitica and avirulent and virulent strains of Pseudomonas syringae were compromised, suggesting that MPK6 plays a role in both resistance gene-mediated and basal resistance. Furthermore, this result demonstrates that MPK6's function cannot be fully complemented by other endogenous MAPKs. Although MPK6-silenced plants exhibited enhanced disease susceptibility, their ability to develop systemic acquired resistance or induced systemic resistance was unaffected. Expression of the pathogen-inducible gene VEGETATIVE STORAGE PROTEIN1 (VSP1) in MPK6-silenced plants was severalfold lower than in control plants, but the expression of other defense genes was comparable to the level observed in control plants. Taken together, these results provide direct evidence that a specific MAPK positively regulates VSP1 expression and resistance to a primary infection by certain pathogens, whereas systemic resistance and expression of several other defense genes appears to be mediated either by a functionally redundant MAPK(s) or independently from MPK6-dependent resistance.     

Systemic resistance in Arabidopsis induced by rhizobacteria requires ethylene-dependent signaling at the site of application.

Root colonization of Arabidopsis thaliana by the nonpathogenic, rhizosphere-colonizing, biocontrol bacterium Pseudomonas fluorescens WCS417r has been shown to elicit induced systemic resistance (ISR) against Pseudomonas syringae pv. tomato (Pst). The ISR response differs from the pathogen-inducible systemic acquired resistance (SAR) response in that ISR is independent of salicylic acid and not associated with pathogenesis-related proteins. Several ethylene-response mutants were tested and showed essentially normal symptoms of Pst infection. ISR was abolished in the ethylene-insensitive mutant etr1-1, whereas SAR was unaffected. Similar results were obtained with the ethylene-insensitive mutants ein2 through ein7, indicating that the expression of ISR requires the complete signal-transduction pathway of ethylene known so far. The induction of ISR by WCS417r was not accompanied by increased ethylene production in roots or leaves, nor by increases in the expression of the genes encoding the ethylene biosynthetic enzymes 1-aminocyclopropane-1-carboxylic (ACC) synthase and ACC oxidase. The eir1 mutant, displaying ethylene insensitivity in the roots only, did not express ISR upon application of WCS417r to the roots, but did exhibit ISR when the inducing bacteria were infiltrated into the leaves. These results demonstrate that, for the induction of ISR, ethylene responsiveness is required at the site of application of inducing rhizobacteria.     

Functional Analysis of Hyaloperonospora arabidopsidis RXLR Effectors

The biotrophic plant pathogen Hyaloperonospora arabidopsidis produces a set of putative effector proteins that contain the conserved RXLR motif. For most of these RXLR proteins the role during infection is unknown. Thirteen RXLR proteins from H. arabidopsidis strain Waco9 were analyzed for sequence similarities and tested for a role in virulence. The thirteen RXLR proteins displayed conserved N-termini and this N-terminal conservation was also found in the 134 predicted RXLR genes from the genome of H. arabidopsidis strain Emoy2. To investigate the effects of single RXLR effector proteins on plant defense responses, thirteen H. arabidopsidis Waco9 RXLR genes were expressed in Arabidopsis thaliana. Subsequently, these plants were screened for altered susceptibility to the oomycetes H. arabidopsidis and Phytophthora capsici, and the bacterial pathogen Pseudomonas syringae. Additionally, the effect of the RXLR proteins on flg22-triggered basal immune responses was assessed. Multifactorial analysis of results collated from all experiments revealed that, except for RXLR20, all RXLR effector proteins tested affected plant immunity. For RXLR9 this was confirmed using a P. syringae ΔCEL-mediated effector delivery system. Together, the results show that many H. arabidopsidis RXLR effectors have small effects on the plant immune response, suggesting that suppression of host immunity by this biotrophic pathogen is likely to be caused by the combined actions of effectors.     

Identification of low abundant secreted proteins and peptides from primary culture supernatants of human T-cells

Live cells continually communicate with their surroundings by the secretion of biomolecules, among which proteins and/or peptides are an important class. As such, these protein/peptide signals which end up in the extracellular medium, reflect the state of a cell in a certain condition, and as by definition are potential biomarkers indicative for specific physiological/pathological processes. We here report on a mass spectrometry based method for the detection and analysis of peptides and proteins secreted in a highly complex background, such as cell culture supernatant. Our method, which combines chromatography, high duty cycle tandem mass spectrometry and bio-informatics, enables the detection of interleukin-2 (IL-2), a cytokine secreted by activated T-cells, present in cell supernatant while representing only 0.006‰ of the total protein content. Moreover, the method allows the mass spectrometric analysis of signaling proteins in a non-targeted way and without any prior immunodepletion of the highest abundant cell culture medium proteins. In this study this is exemplified by the detection of yet two other secretory peptides, i.e., the granulins A and B, in the primary culture supernatant of non-activated T-cells.