Ana Maria Laxalt

The 1988 papers by Mauch, Mauch-Mani, Hadwiger, and Boller on pea antifungal hydrolases captured a simple but powerful idea: plants can attack pathogens by directly targeting the structural polymers that hold their cell walls together. At that time, chitinases and β-1,3-glucanases were known to accumulate after infection, wounding, and ethylene treatment, but whether these proteins actually restricted fungal growth remained unclear. By resolving distinct hydrolase isoforms and showing that inducible chitinase and β-1,3-glucanase act synergistically to inhibit fungal growth and lyse hyphal tips, these studies transformed infection-induced enzyme accumulation into a mechanistic model of antifungal activity. We now recognize that fungal cell wall hydrolysis also releases glycan fragments that act as immune signals and are perceived by plant immune receptors, linking pathogen damage to immune activation in plants. At the same time, pathogens have evolved counterstrategies to mask, modify, or protect these cell wall components, limiting both enzymatic access and immune detection. Thus, the Boller studies anticipated a central theme of modern molecular plant pathology: plant–pathogen interactions are dynamic interfaces where host enzymes, pathogen wall fragments, immune receptors, and pathogen counterstrategies converge.

Phosphoinositide-specific phospholipases C (PI-PLCs) are signaling enzymes that hydrolyze membrane phosphoinositides to generate lipid- and lipid-derived second messengers. In plants, PI-PLCs have been implicated in various physiological processes, including immunity. Tomato SlPLC2 was previously shown to be implicated in susceptibility to the necrotrophic fungus Botrytis cinerea. However, whether this observation extends to pathogens with different lifestyles and evolutionary origins remains unknown. Here, we investigated SlPLC2 function during infection with Phytophthora infestans, a globally devastating oomycete and causal agent of potato and tomato late blight. CRISPR/Cas9 knockout SlPLC2 plants showed significantly reduced disease symptoms, lower pathogen biomass, and decreased sporangia production, indicating impaired colonization. Upon infection, SlPLC2 knockout lines displayed attenuated expression of salicylic acid- and jasmonic acid-marker genes. Consistent with a role for PLC signaling in plant defense, early immune responses, such as hydrogen peroxide accumulation and callose deposition, were reduced in knockout plants. At the cellular level, these plants allow the formation of fewer infection vesicles by P. infestans, accompanied by an increase in expression of a biotrophy-associated effector gene PiAvrblb2, suggesting impaired establishment of infection. In Nicotiana benthamiana, SlPLC2-GFP displays a localization pattern predominately consistent with plasma membrane association and also localizes to the membranes surrounding P. infestans infection vesicles, where we also detected phosphoinositides PI4P and PI(4,5)P2. As complementary heterologous evidence, transient overexpression of SlPLC2 in N. benthamiana enhanced lesion development after P. infestans inoculations. Together, these findings identify SlPLC2 as a susceptibility factor associated with enhanced P. infestans colonization.

Stomata are natural pores through which plants exchange gases with the environment, mainly carbon dioxide and oxygen required for photosynthesis and respiration, as well as water vapor through evapotranspiration. However, they also serve as entry points for microbial pathogens such as Pseudomonas syringae pv. tomato (Pst) bacteria. To prevent microbe invasion, guard cells detect pathogen-associated molecular patterns (PAMPs), including the bacterial peptide flagellin (flg22), triggering stomatal closure. This study identifies hydrogen sulfide (H2S) and its cytosolic source L-CYSTEINE DESULFHYDRASE 1 (DES1) as key players in stomatal immunity. We demonstrate that H2S and DES1 are involved in flg22- and bacterial-induced responses, including stomatal closure and modulation of reactive oxygen species (ROS) production, in Arabidopsis (Arabidopsis thaliana). Knockout mutants defective in the DES1 gene exhibited reduced susceptibility to Pst spray-inoculation and lower apoplastic and cytosolic H2O2 levels in response to flg22. Additionally, H2S independently induced cytosolic H2O2 levels in guard cells without requiring RBOHD activity. These findings establish H2S and its source, DES1, as critical components of the stomatal immune response.

Phosphoinositide-specific phospholipase C (PI-PLC) is a signaling enzyme that hydrolyzes membrane phosphoinositide to generate lipid- and lipid-derived second messengers. In plants, PI-PLCs have been implicated in various physiological processes, including immunity. Tomato SlPLC2 was previously shown to be implicated in susceptibility to the necrotrophic fungus Botrytis cinerea . However, whether this observation extends to pathogens with different lifestyles and evolutionary origins, remains unknown. Here, we investigated SlPLC2 function during infection with Phytophthora infestans , a globally devastating oomycete and causal agent of potato and tomato late blight. CRISPR-Cas9 knockout SlPLC2 plants showed significantly reduced disease symptoms, lower pathogen biomass, and decreased sporangia production, indicating impaired colonization. Upon infection, SlPLC2 knockout lines displayed attenuated expression of salicylic acid (SA)- and jasmonic acid (JA)-responsive genes suggesting disrupted hormone signaling. Consistent with PLC role during plant defense, early immune responses such as hydrogen peroxide accumulation and callose deposition were reduced in knockout plants. At the cellular level, these plants allow fewer infection vesicles formation by P. infestans , accompanied by an increase in expression of a biotrophy-associated effector gene PiAvrblb2 , suggesting impaired establishment of infection. In Nicotiana benthamiana SlPLC2-GFP localizes predominantly at the plasma membrane. Upon P. infestans inoculations, SlPLC2-GFP localizes to the membrane surrounding infection vesicles, where we also detected phosphoinositides PI4P and PI(4,5)P₂. Overexpression of SlPLC2 in Nicotiana benthamiana enhanced susceptibility to P. infestans , reinforcing its positive role in colonization. Together, these findings identify SlPLC2 as a susceptibility factor associated with enhanced P. infestans colonization. Statement: This study identifies SlPLC2 as a key susceptibility factor that supports Phytophthora infestans infection by coordinating vesicle formation and early immune responses in tomato.

Phosphoinositide-specific phospholipase C (PI-PLC) signaling is involved in various plant stress and developmental responses. Though several aspects of this lipid signaling pathway are conserved within animals and plants, clear differences have also emerged. While animal PLC signaling is characterized by the hydrolysis of PIP2 and production of IP3 and DAG as second messengers to activate Ca2+ and PKC signaling, plant PI-PLCs seem to predominantly use PIP as substrate and convert IP2 and DAG into inositolpolyphosphates and phosphatidic acid (PA) as plant second messengers. Sequencing of multiple plant genomes confirmed that plant PLC signaling evolved differently from animals, lacking homologs of the IP3 gated-Ca2+ channel, PKC and TRP channels, and with PLC enzymes resembling the PLCζ subfamily, which lacks the conserved PH domain that binds PIP2. With emerging tools in plant molecular biology, data analyses, and advanced imaging, plant PLC signaling is ready to gain momentum.

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. Abstract 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.

Sulforaphane (SFN) is an isothiocyanate‐type phytomolecule present in crucifers, which is mainly synthesized in response to biotic stress. In animals, SFN incorporated in the diet has anticancer properties among others. The mechanism of action and signaling are well described in animals; however, little is known in plants. The goal in the present study is to elucidate components of the SFN signaling pathway, particularly the production of reactive oxygen species (ROS), and its effect on the transcriptome. Our results showed that in Arabidopsis, SFN causes ROS production exclusively through the action of the NADPH oxidase RBOH isoform D that requires calcium as a signaling component for the ROS production. To add to this, we also analyzed the effect of SFN on the transcriptome by RNAseq. We observed the highest expression increase for heat shock proteins (HSP) genes and also for genes associated with the response to oxidative stress. The upregulation of several genes linked to the biotic stress response confirms the interplay between SFN and this stress. In addition, SFN increases the levels of transcripts related to the response to abiotic stress, as well as phytohormones. Taken together, these results indicate that SFN induces an oxidative burst leading to signaling events. This oxidative burst may cause the increase of the expression of genes such as heat shock proteins to restore cellular homeostasis and genes that codify possible components of the signaling pathway and putative effectors.

Main conclusion Nitro fatty acids (NO2-FA)have relevant physiological roles as signaling molecules in biotic and abiotic stress, growth, and development, but the mechanism of action remains controversial. The two main mechanisms involving nitric oxide release and thiol modification are discussed. Abstract Fatty acids (FAs) are major components of membranes and contribute to cellular energetic demands. Besides, FAs are precursors of signaling molecules, including oxylipins and other oxidized fatty acids derived from the activity of lipoxygenases. In addition, non-canonical modified fatty acids, such as nitro-fatty acids (NO₂-FAs), are formed in animals and plants. The synthesis NO₂-FAs involves a nitration reaction between unsaturated fatty acids and reactive nitrogen species (RNS). This review will focus on recent findings showing that, in plants, NO₂-FAs such as nitro-linolenic acid (NO₂-Ln) and nitro-oleic acid (NO₂-OA) have relevant physiological roles as signaling molecules in biotic and abiotic stress, growth, and development. Moreover, since there is controversy on mechanisms of action of NO₂-FAs as signaling molecules, we will provide evidence showing why this aspect needs further evaluation.

Nitric oxide (NO) is a second messenger that regulates a broad range of physiological processes in plants. NO-derived molecules called reactive nitrogen species (RNS) can react with unsaturated fatty acids generating nitrated fatty acids (NO₂-FA). NO₂-FA work as signaling molecules in mammals where production and targets have been described under different stress conditions. Recently, NO₂-FAs were detected in plants, however their role(s) on plant physiological processes is still poorly known. Although in this work NO2-OA has not been detected in any Arabidopsis seedling tissue, here we show that exogenous application of nitro-oleic acid (NO2-OA) inhibits Arabidopsis primary root growth; this inhibition is not likely due to nitric oxide (NO) production or impaired auxin or cytokinin root responses. Deep analyses showed that roots incubated with NO₂-OA had a lower cell number in the division area. Although this NO₂-FA did not affect the hormonal signaling mechanisms maintaining the stem cell niche, plants incubated with NO₂-OA showed a reduction of cell division in the meristematic area. Therefore, this work shows that the exogenous application of NO₂-OA inhibits mitotic processes subsequently reducing primary root growth.