María Elena Alvarez

Reactive Oxygen Intermediates Mediate a Systemic Signal Network in the Establishment of Plant Immunity

Recognition of an avirulent pathogen stimulates an oxidative burst generating O2- and H2O2, and these reactive oxygen intermediates (ROIs) cue the induction of defense genes and cell death in the development of a restricted lesion. This localized hypersensitive response (HR) is accompanied by the development of systemic acquired resistance to virulent pathogens. Here we show that inoculation of Arabidopsis leaves with avirulent Pseudomonas syringae induces secondary oxidative bursts in discrete cells in distant tissues, leading to low-frequency systemic micro-HRs. The primary oxidative burst induces these systemic responses, and both the primary burst and the secondary microbursts are required for systemic immunity. Hence, ROIs mediate a reiterative signal network underlying systemic as well as local resistance responses.

Calcium-mediated apoptosis in a plant hypersensitive disease resistance response

BACKGROUND Avirulent pathogens elicit a battery of plant defenses, often accompanied by collapse of the challenged cells. In soybean cells, sustained accumulation of H2O2 from an oxidative burst cues localized host cell death. Such hypersensitive cell death appears to be an active process, but little is known about the mechanisms underlying cellular collapse. RESULTS We show that H2O2 stimulates a rapid influx of Ca2+ into soybean cells, which activates a physiological cell death program resulting in the generation of large (approximately 50 kb) DNA fragments and cell corpse morphology--including cell shrinkage, plasma membrane blebbing and nuclear condensation--characteristic of apoptosis. In contrast, H2O2 induction of the cellular protectant gene glutathione S-transferase is Ca(2+)-independent. Apoptosis in soybean cells and leaf tissue was induced by avirulent Pseudomonas syringae pv. glycinea but was not observed at comparable stages of the compatible interaction with the isogenic virulent strain, which fails to elicit a hypersensitive response. Apoptosis was also observed at the onset of the hypersensitive response in Arabidopsis leaves inoculated with avirulent P. syringae pv. tomato and in tobacco cells treated with the fungal peptide cryptogein, which is involved in the induction of non-host resistance to Phytophthora cryptogea. CONCLUSIONS These observations establish a signal function for Ca2+ downstream of the oxidative burst in the activation of a physiological cell death program in soybean cells that is similar to apoptosis in animals. That the characteristic cell corpse morphology is also induced in Arabidopsis and tobacco by different avirulence signals suggests that apoptosis may prove to be a common, but not necessarily ubiquitous, feature of incompatible plant-pathogen interactions. Emerging similarities between facets of hypersensitive disease resistance and the mammalian native immune system indicate that apoptosis is a widespread defence mechanism in eukaryotes.

Salicylic acid in the machinery of hypersensitive cell death and disease resistance

Although extensive data has described the key role of salicylic acid (SA) in signaling pathogen-induced disease resistance, its function in physiological processes related to cell death is still poorly understood. Recent studies have explored the requirement of SA for mounting the hypersensitive response (HR) against an invading pathogen, where a particular cell death process is activated at the site of attempted infection causing a confined lesion. Biochemical data suggest that SA potentiates the signal pathway for HR by affecting an early phosphorylation-sensitive step preceding the generation of pro-death signals, including those derived from the oxidative burst. Accordingly, the epistatic relationship between cell death and SA accumulation, analyzed in crosses between lesion-mimic mutants (spontaneous lesion formation) and the transgenic nahG line (depleted in SA) places the SA activity in a feedback loop downstream and upstream of cell death. Exciting advances have been made in the identification of cellular protective functions and cell death suppressors that might operate in HR. Moreover, the spatio-temporal patterns of the SA accumulation (non-homogeneous distribution, biphasic kinetics) described in some HR lesions, may also reveal important clues for unraveling the complex cellular network that tightly balances pro- and anti-death functions in the hypersensitive cell death.

Ascorbic Acid Deficiency Activates Cell Death and Disease Resistance Responses in Arabidopsis

Programmed cell death, developmental senescence, and responses to pathogens are linked through complex genetic controls that are influenced by redox regulation. Here we show that the Arabidopsis (Arabidopsis thaliana) low vitamin C mutants, vtc1 and vtc2, which have between 10% and 25% of wild-type ascorbic acid, exhibit microlesions, express pathogenesis-related (PR) proteins, and have enhanced basal resistance against infections caused by Pseudomonas syringae. The mutants have a delayed senescence phenotype with smaller leaf cells than the wild type at maturity. The vtc leaves have more glutathione than the wild type, with higher ratios of reduced glutathione to glutathione disulfide. Expression of green fluorescence protein (GFP) fused to the nonexpressor of PR protein 1 (GFP-NPR1) was used to detect the presence of NPR1 in the nuclei of transformed plants. Fluorescence was observed in the nuclei of 6- to 8-week-old GFP-NPR1 vtc1 plants, but not in the nuclei of transformed GFP-NPR1 wild-type plants at any developmental stage. The absence of senescence-associated gene 12 (SAG12) mRNA at the time when constitutive cell death and basal resistance were detected confirms that elaboration of innate immune responses in vtc plants does not result from activation of early senescence. Moreover, H2O2-sensitive genes are not induced at the time of systemic acquired resistance execution. These results demonstrate that ascorbic acid abundance modifies the threshold for activation of plant innate defense responses via redox mechanisms that are independent of the natural senescence program.

Proline accumulation and AtP5CS2 gene activation are induced by plant-pathogen incompatible interactions in Arabidopsis.

Accumulation of free L-proline (Pro) is a typical stress response incited by osmotic injuries in plants and microorganisms. Although the protective role of Pro in osmotic stress is not well understood, it is thought to function as compatible osmolyte or as a scavenger of reactive oxygen species (ROS). Here we show that, in Arabidopsis thaliana, Pro biosynthesis can be activated by incompatible plant-pathogen interactions triggering a hypersensitive response (HR). Pro accumulates in leaf tissues treated with Pseudomonas syringae pv. tomato avirulent strains (avrRpt2 and avrRpm1) but remains unchanged in leaves infected with isogenic virulent bacteria. Incompatible interactions lead to transcriptional activation of AtP5CS2, but not AtP5CS1, encoding the rate limiting enzyme in Pro biosynthesis pyrroline-5-carboxylate synthase (P5CS). AtP5CS2:GUS and AtP5CS2:LUC transgenes were induced inside and around the HR lesions produced by avirulent Pseudomonas spp. in transgenic plants. Pro accumulation was faster and stronger when stimulated by avrRpm1 than by avrRpt2, and was compromised in the low-salicylic acid plants NahG and eds5 when signaled through the RPS2-dependent pathway. In addition, Pro content and AtP5CS2 expression were enhanced by ROS in wild-type plants, suggesting that ROS may function as an intermediate signal in AtP5CS2-mediated Pro accumulation.

Early genomic responses to salicylic acid in Arabidopsis

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.

Proline Dehydrogenase Contributes to Pathogen Defense in Arabidopsis

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.

Genome-Wide Expression Profiling Arabidopsis at the Stage of Golovinomyces cichoracearum Haustorium Formation

Compatibility between plants and obligate biotrophic fungi requires fungal mechanisms for efficiently obtaining nutrients and counteracting plant defenses under conditions that are expected to induce changes in the host transcriptome. A key step in the proliferation of biotrophic fungi is haustorium differentiation. Here we analyzed global gene expression patterns in Arabidopsis thaliana leaves during the formation of haustoria by Golovinomyces cichoracearum. At this time, the endogenous levels of salicylic acid (SA) and jasmonic acid (JA) were found to be enhanced. The responses of wild-type, npr1-1, and jar1-1 plants were used to categorize the sensitivity of gene expression changes to NPR1 and JAR1, which are components of the SA and JA signaling pathways, respectively. We found that the infection process was the major source of variation, with 70 genes identified as having similarly altered expression patterns regardless of plant genotype. In addition, principal component analysis (PCA) identified genes responding both to infection and to lack of functional JAR1 (17 genes) or NPR1 (18 genes), indicating that the JA and SA signaling pathways function as secondary sources of variation. Participation of these genes in the SA or JA pathways had not been described previously. We found that some of these genes may be sensitive to the balance between the SA and JA pathways, representing novel markers for the elucidation of cross-talk points between these signaling cascades. Conserved putative regulatory motifs were found in the promoter regions of each subset of genes. Collectively, our results indicate that gene expression changes in response to infection by obligate biotrophic fungi may support fungal nutrition by promoting alterations in host metabolism. In addition, these studies provide novel markers for the characterization of defense pathways and susceptibility features under this infection condition.

Mycobacterial Ecology of the Rio Grande

ABSTRACT This is the first study to characterize the environmental conditions which contribute to the presence and proliferation of environmental mycobacteria in a major freshwater river. Over 20 different species of environmental mycobacteria were isolated, including the pathogenic M. avium and M. kansasii. Species of the rapidly growing M. fortuitum complex were the most commonly isolated mycobacteria, and one-third of all isolates were not identified at the species level, even by 16S sequencing. PCR restriction analysis of the hsp65 gene was more accurate and rapid than biochemical tests and as accurate as yet less expensive than 16S sequencing, showing great promise as a new tool for species identification of environmentally isolated mycobacteria. Total environmental mycobacteria counts positively correlated with coliform and Escherichia coli counts and negatively correlated with chemical toxicity and water temperature. Environmental mycobacteria can survive in the alkaline conditions of the river despite previous reports that especially acidic conditions favor their presence. A representative river isolate (M. fortuitum) survived better than E. coli O157:H7 at pHs below 7 and above 8 in nutrient broth. The river strain also retained viability at 8 ppm of free chlorine, while E. coli was eliminated at 2 ppm and above. Thus, in vitro studies support environmental observations that a variety of extreme conditions favor the hardy environmental mycobacteria.

Histone H1 Is Required for Proper Regulation of Pyruvate Decarboxylase Gene Expression in Neurospora crassa

ABSTRACT We show that Neurosporacrassa has a single histone H1 gene, hH1, which encodes a typical linker histone with highly basic N- and C-terminal tails and a central globular domain. A green fluorescent protein-tagged histone H1 chimeric protein was localized exclusively to nuclei. Mutation of hH1 by repeat-induced point mutation (RIP) did not result in detectable defects in morphology, DNA methylation, mutagen sensitivity, DNA repair, fertility, RIP, chromosome pairing, or chromosome segregation. Nevertheless, hH1 mutants had mycelial elongation rates that were lower than normal on all tested carbon sources. This slow linear growth phenotype, however, was less evident on medium containing ethanol. The pyruvate decarboxylase gene, cfp, was abnormally derepressed in hH1 mutants on ethanol-containing medium. This derepression was also found when an ectopically integrated fusion of the cfp gene promoter to the reporter gene hph was analyzed. Thus, Neurospora histone H1 is required for the proper regulation of cfp, a gene with a key role in the respiratory-fermentative pathway.