Loss-of-function mutations in WRKY22 and WRKY25 impair stomatal-mediated immunity and PTI and ETI responses against Pseudomonas syringae pv. tomato.

Plants defend themselves against pathogens using a two-layered immune system. The first response, pattern-triggered immunity (PTI), is activated upon recognition of microbe-associated molecular patterns (MAMPs). Virulent bacteria such as Pseudomonas syringae pv. tomato (Pst), deliver effector proteins into the plant cell to promote susceptibility. However, some plants possess resistance (R) proteins that recognize specific effectors leading to the activation of the second response, effector-triggered immunity (ETI). Resistant tomatoes such as Río Grande-PtoR recognize two Pst effectors (AvrPto and AvrPtoB) through the host Pto/Prf complex and activate ETI. We previously showed that the transcription factors (TF) WRKY22 and WRKY25 are positive regulators of plant immunity against bacterial and potentially non-bacterial pathogens in Nicotiana benthamiana. Here, the CRISPR-Cas9 technique was used to develop three knockout tomato lines for either one or both TFs. The single and double mutants were all compromised in Pto/Prf-mediated ETI and had a weaker PTI response. The stomata apertures in all of the mutant lines did not respond to darkness or challenge with Pst DC3000. The WRKY22 and WRKY25 proteins both localize in the nucleus, but we found no evidence of a physical interaction between them. The WRKY22 TF was found to be involved in the transcriptional regulation of WRKY25, supporting the idea that they are not functionally redundant. Together, our results indicate that both WRKY TFs play a role in modulating stomata and are positive regulators of plant immunity in tomato.

Variability in flooding tolerance, growth and leaf traits in a Populus deltoides intraspecific progeny.

Climate change will increase the risk of flooding in several areas of the world where Populus deltoides Marshall (eastern cottonwood) is planted, so it would be desirable for this species to select for flooding tolerance. The aims of this work were to explore the variability in growth, leaf traits and flooding tolerance in an F1 full-sib intraspecific progeny of P. deltoides, to analyze the correlations of leaf and growth traits with flooding tolerance and to assess their suitability for use in breeding programs. Two-month-old parental clones and their progeny of 30 full-sib F1 genotypes were grown in pots and subjected to two treatments: (i) plants watered to field capacity (control) and (ii) plants flooded up to 10 cm above soil level for 35 days. Growth (height, diameter and biomass partition) and leaf traits (leaf size and number, specific leaf area, leaf senescence, abscission, stomatal conductance, carbon isotope discrimination, stomatal index) were measured. Flooding tolerance for each genotype was estimated as the ratio of the biomass of stressed plants to the biomass of control plants. Results showed segregation in terms of flooding tolerance in the F1 progeny. A significant genotype effect was found for leaf size and number, carbon isotopic discrimination and stomatal conductance, but it did not correlate with flooding tolerance. Height, diameter and root-to-shoot ratio had a positive phenotypic correlation with flooding tolerance, and there was a positive genetic correlation of height and diameter with biomass on both treatments. The narrow sense heritability values for the traits analyzed ranged from 0 to 0.56. We conclude that growth traits are more adequate than leaf traits for selection to increase flooding tolerance. A vigorous initial growth would increase flooding tolerance in young poplar plants.

Potassium deprivation is sufficient to induce a cell death program in Saccharomyces cerevisiae.

Cell culture in low potassium (K(+)) media has been associated to programmed cell death (PCD) in metazoans. In this study, deprivation of K(+) led Saccharomyces cerevisiae cells to a death process that involved phosphatidylserine externalization, changes in chromatin condensation, DNA and vacuole fragmentation as well as enhanced accumulation of reactive oxygen species. During the course of K(+) starvation, plasma membrane hyperpolarization and increased accumulation of calcium (Ca(2+)) took place. The presence of rubidium (Rb(+)), a K(+)-analogue element, in the K(+)-deprived medium was accompanied by Rb(+) accumulation but did not fully prevent the appearance of PCD markers. This argues for a specific effect of K(+) on the course of cell death. While the absence of the YCA1 metacaspase did not have a major effect, the absence of TRK (transport of K(+)) K(+)-transporters led to changes in the pattern of annexin V/propidium iodide labeling. This change paralleled a fast accumulation of Ca(2+). Addition of ethylene glycol tetraacetic acid improved growth and reduced cell death in trk1Deltatrk2Delta cells. These findings reveal that K(+) deprivation is sufficient to induce PCD in a cell-walled eukaryotic organism and suggest that the phenotype attributed to the lack of TRK genes is partially due to the effect of the encoded transporters on Ca(2+) homeostasis.