Fighting Tomato Fungal Diseases with a Biocontrol Product Based on Amoeba Lysate.

New solutions to reduce the use of chemical pesticides to combat plant diseases and to meet societal and political demands are needed to achieve sustainable agriculture. Tomato production, both in greenhouses and in open fields, is affected by numerous pathogens. The aim of this study is to assess the possibility of controlling both late blight and powdery mildew in tomatoes with a single biocontrol product currently under registration. The biocontrol product AXP12, based on the lysate of Willaertia magna C2c Maky, has already proved its efficacy against downy mildew of grapevine and potato late blight. Its ability to elicit tomato defenses and its efficacy in the greenhouse and in the field were tested. This study establishes that AXP12 stimulates the tomato genes involved in plant defense pathways and has the capacity to combat in greenhouse and field both late blight (Phytophtora infestans) and powdery mildew (Oidium neolycopersici and Leveillula taurica) of tomato.

A New Biocontrol Tool to Fight Potato Late Blight Based on Willaertia magna C2c Maky Lysate.

Potato late blight (PLB) is one of the most destructive disease affecting potatoes. Late blight control relies almost exclusively on the use of chemical pesticides, including copper products, which are efficient but controversial due to their environmental toxicity. Societal pressure and the quest for more sustainable agriculture reinforce the need for natural plant protection products. To respond to this demand, we tested the lysate of the amoeba Willaertia magna C2c Maky on PLB. This active substance exhibits plant protection properties against grape downy mildew thanks to a dual mode of action (plant elicitor and antifungal direct effect). We hypothesized that this active substance might also have an effect against other diseases caused by oomycetes on other crops, such as potato. In vitro, in planta, and in-field studies were conducted. The collected data demonstrate that the lysate of the amoeba Willaertia magna C2c Maky is able to elicit potato defenses, and direct fungicidal activity against Phytophtora infestans was observed. Proof of efficacy was first obtained in greenhouse, with up to 80% disease reduction, and confirmed in field trials. Formulated products provided up to 77% protection in field in the case of low infestation (28%) and up to 49% protection when the untreated plants were 100% destroyed. Willaertia magna C2c Maky was also able to significantly increase yield by up to 30% in field trials.

Dual Targeting of the Protein Methyltransferase PrmA Contributes to Both Chloroplastic and Mitochondrial Ribosomal Protein L11 Methylation in Arabidopsis.

Methylation of ribosomal proteins has long been described in prokaryotes and eukaryotes, but our knowledge about the enzymes responsible for these modifications in plants is scarce. The bacterial protein methyltransferase PrmA catalyzes the trimethylation of ribosomal protein L11 (RPL11) at three distinct sites. The role of these modifications is still unknown. Here, we show that PrmA from Arabidopsis thaliana (AtPrmA) is dually targeted to chloroplasts and mitochondria. Mass spectrometry and enzymatic assays indicated that the enzyme methylates RPL11 in plasto- and mitoribosomes in vivo. We determined that the Arabidopsis and Escherichia coli PrmA enzymes share similar product specificity, making trimethylated residues, but, despite an evolutionary relationship, display a difference in substrate site specificity. In contrast to the bacterial enzyme that trimethylates the ε-amino group of two lysine residues and the N-terminal α-amino group, AtPrmA methylates only one lysine in the MAFCK(D/E)(F/Y)NA motif of plastidial and mitochondrial RPL11. The plant enzyme possibly methylates the N-terminus of plastidial RPL11, whereas mitochondrial RPL11 is N-α-acetylated by an unknown acetyltransferase. Lastly, we found that an Arabidopsis prma-null mutant is viable in standard environmental conditions and no molecular defect could be associated with a lack of RPL11 methylation in leaf chloroplasts or mitochondria. However, the conservation of PrmA during the evolution of photosynthetic eukaryotes together with the location of methylated residues at the binding site of translation factors to ribosomes suggests that RPL11 methylation in plant organelles could be involved, in combination with other post-translational modifications, in optimizing ribosome function.

Uncovering the protein lysine and arginine methylation network in Arabidopsis chloroplasts.

Post-translational modification of proteins by the addition of methyl groups to the side chains of Lys and Arg residues is proposed to play important roles in many cellular processes. In plants, identification of non-histone methylproteins at a cellular or subcellular scale is still missing. To gain insights into the extent of this modification in chloroplasts we used a bioinformatics approach to identify protein methyltransferases targeted to plastids and set up a workflow to specifically identify Lys and Arg methylated proteins from proteomic data used to produce the Arabidopsis chloroplast proteome. With this approach we could identify 31 high-confidence Lys and Arg methylation sites from 23 chloroplastic proteins, of which only two were previously known to be methylated. These methylproteins are split between the stroma, thylakoids and envelope sub-compartments. They belong to essential metabolic processes, including photosynthesis, and to the chloroplast biogenesis and maintenance machinery (translation, protein import, division). Also, the in silico identification of nine protein methyltransferases that are known or predicted to be targeted to plastids provided a foundation to build the enzymes/substrates relationships that govern methylation in chloroplasts. Thereby, using in vitro methylation assays with chloroplast stroma as a source of methyltransferases we confirmed the methylation sites of two targets, plastid ribosomal protein L11 and the ß-subunit of ATP synthase. Furthermore, a biochemical screening of recombinant chloroplastic protein Lys methyltransferases allowed us to identify the enzymes involved in the modification of these substrates. The present study provides a useful resource to build the methyltransferases/methylproteins network and to elucidate the role of protein methylation in chloroplast biology.

Characterization of chloroplastic fructose 1,6-bisphosphate aldolases as lysine-methylated proteins in plants.

In pea (Pisum sativum), the protein-lysine methyltransferase (PsLSMT) catalyzes the trimethylation of Lys-14 in the large subunit (LS) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme catalyzing the CO(2) fixation step during photosynthesis. Homologs of PsLSMT, herein referred to as LSMT-like enzymes, are found in all plant genomes, but methylation of LS Rubisco is not universal in the plant kingdom, suggesting a species-specific protein substrate specificity of the methyltransferase. In this study, we report the biochemical characterization of the LSMT-like enzyme from Arabidopsis thaliana (AtLSMT-L), with a focus on its substrate specificity. We show that, in Arabidopsis, LS Rubisco is not naturally methylated and that the physiological substrates of AtLSMT-L are chloroplastic fructose 1,6-bisphosphate aldolase isoforms. These enzymes, which are involved in the assimilation of CO(2) through the Calvin cycle and in chloroplastic glycolysis, are trimethylated at a conserved lysyl residue located close to the C terminus. Both AtLSMT-L and PsLSMT are able to methylate aldolases with similar kinetic parameters and product specificity. Thus, the divergent substrate specificity of LSMT-like enzymes from pea and Arabidopsis concerns only Rubisco. AtLSMT-L is able to interact with unmethylated Rubisco, but the complex is catalytically unproductive. Trimethylation does not modify the kinetic properties and tetrameric organization of aldolases in vitro. The identification of aldolases as methyl proteins in Arabidopsis and other species like pea suggests a role of protein lysine methylation in carbon metabolism in chloroplasts.