ISHS VI International Solanaceae Conference : Genomics Meets Biodiversity THE EVOLUTION OF CHILI PEPPERS (CAPSICUM - SOLANACEAE): A CYTOGENETIC PERSPECTIVE

L-Proline (Pro) catabolism is activated in plants recovering from abiotic stresses associated with water deprivation. In this catabolic pathway, Pro is converted to glutamate by two reactions catalyzed by proline dehydrogenase (ProDH) and Δ¹-pyrroline-5-carboxylate dehydrogenase (P5CDH), with Δ¹-pyrroline-5-carboxylate (P5C) as the intermediate. Alternatively, under certain conditions, the P5C derived from Pro is converted back to Pro by P5C reductase, thus stimulating the Pro-P5C cycle, which may generate reactive oxygen species (ROS) as a consequence of the ProDH activity. We previously observed that Pro biosynthesis is altered in Arabidopsis (Arabidopsis thaliana) tissues that induce the hypersensitive response (HR) in response to Pseudomonas syringae. In this work, we characterized the Pro catabolic pathway and ProDH activity in this model. Induction of ProDH expression was found to be dependent on salicylic acid, and an increase in ProDH activity was detected in cells destined to die. To evaluate the role of ProDH in the HR, ProDH-silenced plants were generated. These plants displayed reduced ROS and cell death levels as well as enhanced susceptibility in response to avirulent pathogens. Interestingly, the early activation of ProDH was accompanied by an increase in P5C reductase but not in P5CDH transcripts, with few changes occurring in the Pro and P5C levels. Therefore, our results suggest that in wild-type plants, ProDH is a defense component contributing to HR and disease resistance, which apparently potentiates the accumulation of ROS. The participation of the Pro-P5C cycle in the latter response is discussed.

Plant tissues display major alterations upon the perception of microbial pathogens. Changes of cytoplasmic and apo-plastic components that sense and transduce plant defenses have been extensively characterized. In contrast, less information is available about modifications affecting the plant nuclear genome under these circumstances. Here, we investigated whether the Arabidopsis thaliana DNA methylation status is altered in tissues responding to the attack of Pseudomonas syringae pv. tomato DC3000. We applied amplified fragment length polymorphism analysis to monitor cytosine methylation at anonymous 5′-CCGG-3′ and 5′-GATC-3′ sites in naïve and infected samples. Plant genomic fragments reducing methylation upon infection, including peri/centromeric repeats such as the 180-bp unit, Athila retrotansposon, and a portion of the nuclear insertion of mitochondrial DNA, were isolated and characterized. P. syringae pv. tomato-induced hypomethylation was detected by high-performance liquid chromatography assays and at the molecular level it did not seem to equally affect all 5-methyl cytosine (5-mC) residues. Nuclei from challenged tissues displayed structural chromatin alterations, including loosening of chromocenters, which also were stimulated by avirulent P. syringae pv. tomato, but not by the P. syringae pv. tomato hrpL¯ mutant. Finally, P. syringae pv. tomato-induced hypomethylation was found to occur in the absence of DNA replication, suggesting that it involves an active demethylation mechanism. All these responses occurred at 1 day postinfection, largely preceding massive plant cell death generated by pathogen attack.

Salicylic acid (SA) is a stress-induced hormone involved in the activation of defense genes. Here we analyzed the early genetic responses to SA of wild type and npr1-1 mutant Arabidopsis seedlings, using Complete Arabidopsis Transcriptome MicroArray (CATMAv2) chip. We identified 217 genes rapidly induced by SA (early SAIGs); 193 by a NPR1-dependent and 24 by a NPR1-independent pathway. These two groups of genes also differed in their functional classification, expression profiles and over-representation of cis-elements, supporting differential pathways for their activation. Examination of the expression patterns for selected early SAIGs from both groups indicated that their activation by SA required TGA2/5/6 subclass of transcription factors. These genes were also activated by Pseudomonas syringae pv. tomato AvrRpm1, suggesting that they might play a role in defense against bacteria. This study gives a global idea of the early response to SA in Arabidopsis seedlings, expanding our knowledge about SA function in plant defense.

Priming is a major mechanism behind the immunological ‘memory’ observed during two key plant systemic defences: systemic acquired resistance (SAR) and induced systemic resistance (ISR). Lipid-derived azelaic acid (AZA) is a mobile priming signal. Here, we show that the lipid transfer protein (LTP)-like AZI1 and its closest paralog EARLI1 are necessary for SAR, ISR and the systemic movement and uptake of AZA in Arabidopsis. Imaging and fractionation studies indicate that AZI1 and EARLI1 localize to expected places for lipid exchange/movement to occur. These are the ER/plasmodesmata, chloroplast outer envelopes and membrane contact sites between them. Furthermore, these LTP-like proteins form complexes and act at the site of SAR establishment. The plastid targeting of AZI1 and AZI1 paralogs occurs through a mechanism that may enable/facilitate their roles in signal mobilization.

A central mechanism of virulence of extracellular bacterial pathogens is the injection into host cells of effector proteins that modify host cellular functions. HopW1 is an effector injected by the type III secretion system that increases the growth of the plant pathogen Pseudomonas syringae on the Columbia accession of Arabidopsis. When delivered by P. syringae into plant cells, HopW1 causes a reduction in the filamentous actin (F-actin) network and the inhibition of endocytosis, a known actin-dependent process. When directly produced in plants, HopW1 forms complexes with actin, disrupts the actin cytoskeleton and inhibits endocytosis as well as the trafficking of certain proteins to vacuoles. The C-terminal region of HopW1 can reduce the length of actin filaments and therefore solubilize F-actin in vitro. Thus, HopW1 acts by disrupting the actin cytoskeleton and the cell biological processes that depend on actin, which in turn are needed for restricting P. syringae growth in Arabidopsis. Eukaryotic cells require a dynamic actin cytoskeleton for basic functions, some of which are important for immune responses. Such functions include the transport of cellular material to and from different cellular compartments. The plant pathogen Pseudomonas syringae is extracellular and causes disease by injecting effector proteins into plant cells. One of these effectors is HopW1, which disrupts the actin cytoskeleton and reduces the transport of vesicles from the cell surface and proteins destined for vacuoles. The effects of HopW1 can be mimicked using a drug that inhibits actin polymerization. Thus, this work establishes a direct mechanism for pathogen disruption of the actin cytoskeleton and implicates actin-dependent events as important for controlling pathogen growth during infection.