CRISPR/Cas9-mediated phospholipase C 2 knock-out tomato plants are more resistant to Botrytis cinerea.

MAIN CONCLUSION: CRISPR/Cas9-mediated Phospholipase C2 knock-out tomato plants are more resistant to Botrytis cinerea than wild-type plants, with less ROS and an increase and reduction of (JA) and (SA)-response marker genes, respectively. Genome-editing technologies allow non-transgenic site-specific mutagenesis of crops, offering a viable alternative to traditional breeding methods. In this study we used CRISPR/Cas9 to inactivate the tomato Phospholipase C2 gene (SlPLC2). Plant PLC activation is one of the earliest responses triggered by different pathogens regulating plant responses that, depending on the plant-pathogen interaction, result in plant resistance or susceptibility. The tomato (Solanum lycopersicum) PLC gene family has six members, named from SlPLC1 to SlPLC6. We previously showed that SlPLC2 transcript levels increased upon xylanase infiltration (fungal elicitor) and that SlPLC2 participates in plant susceptibility to Botrytis cinerea. An efficient strategy to control diseases caused by pathogens is to disable susceptibility genes that facilitate infection. We obtained tomato SlPLC2-knock-out lines with decreased ROS production upon B. cinerea challenge. Since this fungus requires ROS-induced cell death to proliferate, SlPLC2-knock-out plants showed an enhanced resistance with smaller necrotic areas and reduced pathogen proliferation. Thus, we obtained SlPLC2 loss-of-function tomato lines more resistant to B. cinerea by means of CRISPR/Cas9 genome editing technology.

Nitro-oleic acid triggers ROS production via NADPH oxidase activation in plants: A pharmacological approach.

Nitrated fatty acids (NO2-FAs) are important signaling molecules in mammals. NO2-FAs are formed by the addition reaction of nitric oxide- and nitrite-derived nitrogen dioxide with unsaturated fatty acid double bonds. The study of NO2-FAs in plant systems constitutes an interesting and emerging area. The presence of NO2-FA has been reported in olives, peas, rice and Arabidopsis. To gain a better understanding of the role of NO2-FA on plant physiology, we analyzed the effects of exogenous application of nitro-oleic acid (NO2-OA). In tomato cell suspensions we found that NO2-OA induced reactive oxygen species (ROS) production in a dose-dependent manner via activation of NADPH oxidases, a mechanism that requires calcium entry from the extracellular compartment and protein kinase activation. In tomato and Arabidopsis leaves, NO2-OA treatments induced two waves of ROS production, resembling plant defense responses. Arabidopsis NADPH oxidase mutants showed that NADPH isoform D (RBOHD) was required for NO2-OA-induced ROS production. In addition, on Arabidopsis isolated epidermis, NO2-OA induced stomatal closure via RBOHD and F. Altogether, these results indicate that NO2-OA triggers NADPH oxidase activation revealing a new signaling role in plants.

The sesquiterpene botrydial from Botrytis cinerea induces phosphatidic acid production in tomato cell suspensions.

MAIN CONCLUSION: The phytotoxin botrydial triggers PA production in tomato cell suspensions via PLD and PLC/DGK activation. PLC/DGK-derived PA is partially required for botrydial-induced ROS generation. Phosphatidic acid (PA) is a phospholipid second messenger involved in the induction of plant defense responses. It is generated via two distinct enzymatic pathways, either via phospholipase D (PLD) or by the sequential action of phospholipase C and diacylglycerol kinase (PLC/DGK). Botrydial is a phytotoxic sesquiterpene generated by the necrotrophic fungus Botrytis cinerea that induces diverse plant defense responses, such as the production of reactive oxygen species (ROS). Here, we analyzed PA and ROS production and their interplay upon botrydial treatments, employing tomato (Solanum lycopersicum) cell suspensions as a model system. Botrydial induces PA production within minutes via PLD and PLC/DGK. Either inhibition of PLC or DGK diminishes ROS generation triggered by botrydial. This indicates that PLC/DGK is upstream of ROS production. In tomato, PLC is encoded by a multigene family constituted by SlPLC1-SlPLC6 and the pseudogene SlPLC7. We have shown that SlPLC2-silenced plants have reduced susceptibility to B. cinerea. In this work, we studied the role of SlPLC2 on botrydial-induced PA production by silencing the expression of SlPLC2 via a specific artificial microRNA. Upon botrydial treatments, SlPLC2-silenced-cell suspensions produce PA levels similar to wild-type cells. It can be concluded that PA is a novel component of the plant responses triggered by botrydial.

Phospholipase C2 Affects MAMP-Triggered Immunity by Modulating ROS Production.

The activation of phosphoinositide-specific phospholipase C (PI-PLC) is one of the earliest responses triggered by the recognition of several microbe-associated molecular patterns (MAMPs) in plants. The Arabidopsis (Arabidopsis thaliana) PI-PLC gene family is composed of nine members. Previous studies suggested a role for PLC2 in MAMP-triggered immunity, as it is rapidly phosphorylated in vivo upon treatment with the bacterial MAMP flg22. Here, we analyzed the role of PLC2 in plant immunity using an artificial microRNA to silence PLC2 expression in Arabidopsis. We found that PLC2-silenced plants are more susceptible to the type III secretion system-deficient bacterial strain Pseudomonas syringae pv tomato (Pst) DC3000 hrcC- and to the nonadapted pea (Pisum sativum) powdery mildew Erysiphe pisi However, PLC2-silenced plants display normal susceptibility to virulent (Pst DC3000) and avirulent (Pst DC3000 AvrRPM1) P. syringae strains, conserving typical hypersensitive response features. In response to flg22, PLC2-silenced plants maintain wild-type mitogen-activated protein kinase activation and PHI1, WRKY33, and FRK1 immune marker gene expression but have reduced reactive oxygen species (ROS)-dependent responses such as callose deposition and stomatal closure. Accordingly, the generation of ROS upon flg22 treatment is compromised in the PLC2-defficient plants, suggesting an effect of PLC2 in a branch of MAMP-triggered immunity and nonhost resistance that involves early ROS-regulated processes. Consistently, PLC2 associates with the NADPH oxidase RBOHD, suggesting its potential regulation by PLC2.

Arabidopsis phosphatidylinositol-phospholipase C2 (PLC2) is required for female gametogenesis and embryo development.

MAIN CONCLUSION: AtPLC2 is an essential gene in Arabidopsis, since it is required for female gametogenesis and embryo development. AtPLC2 might play a role in cell division during embryo-sac development and early embryogenesis. Phosphoinositide-specific phospholipase C (PI-PLC) plays an important role in signal transduction during plant development and in the response to various biotic- and abiotic stresses. The Arabidopsis PI-PLC gene family is composed of nine members, named PLC1 to PLC9. Here, we report that PLC2 is involved in female gametophyte development and early embryogenesis. Using two Arabidopsis allelic T-DNA insertion lines with different phenotypic penetrations, we observed both female gametophytic defects and aberrant embryos. For the plc2-1 mutant (Ws background), no homozygous plants could be recovered in the offspring from self-pollinated plants. Nonetheless, plc2-1 hemizygous mutants are affected in female gametogenesis, showing embryo sacs arrested at early developmental stages. Allelic hemizygous plc2-2 mutant plants (Col-0 background) present reduced seed set and embryos arrested at the pre-globular stage with abnormal patterns of cell division. A low proportion (0.8%) of plc2-2 homozygous mutants was found to escape lethality and showed morphological defects and disrupted megagametogenesis. PLC2-promoter activity was observed during early megagametogenesis, and after fertilization in the embryo proper. Immunolocalization studies in early stage embryos revealed that PLC2 is restricted to the plasma membrane. Altogether, these results establish a role for PLC2 in both reproductive- and embryo development, presumably by controlling mitosis and/or the formation of cell-division planes.