Differential Expression of Fungal Genes Determines the Lifestyle of Plectosphaerella Strains During Arabidopsis thaliana Colonization.

The fungal genus Plectosphaerella comprises species and strains with different lifestyles on plants, such as P. cucumerina, which has served as model for the characterization of Arabidopsis thaliana basal and nonhost resistance to necrotrophic fungi. We have sequenced, annotated, and compared the genomes and transcriptomes of three Plectosphaerella strains with different lifestyles on A. thaliana, namely, PcBMM, a natural pathogen of wild-type plants (Col-0), Pc2127, a nonpathogenic strain on Col-0 but pathogenic on the immunocompromised cyp79B2 cyp79B3 mutant, and P0831, which was isolated from a natural population of A. thaliana and is shown here to be nonpathogenic and to grow epiphytically on Col-0 and cyp79B2 cyp79B3 plants. The genomes of these Plectosphaerella strains are very similar and do not differ in the number of genes with pathogenesis-related functions, with the exception of secreted carbohydrate-active enzymes (CAZymes), which are up to five times more abundant in the pathogenic strain PcBMM. Analysis of the fungal transcriptomes in inoculated Col-0 and cyp79B2 cyp79B3 plants at initial colonization stages confirm the key role of secreted CAZymes in the necrotrophic interaction, since PcBMM expresses more genes encoding secreted CAZymes than Pc2127 and P0831. We also show that P0831 epiphytic growth on A. thaliana involves the transcription of specific repertoires of fungal genes, which might be necessary for epiphytic growth adaptation. Overall, these results suggest that in-planta expression of specific sets of fungal genes at early stages of colonization determine the diverse lifestyles and pathogenicity of Plectosphaerella strains.

Non-branched ß-1,3-glucan oligosaccharides trigger immune responses in Arabidopsis.

Fungal cell walls, which are essential for environmental adaptation and host colonization by the fungus, have been evolutionarily selected by plants and animals as a source of microbe-associated molecular patterns (MAMPs) that, upon recognition by host pattern recognition receptors (PRRs), trigger immune responses conferring disease resistance. Chito-oligosaccharides [ß-1,4-N-acetylglucosamine oligomers, (GlcNAc)n ] are the only glycosidic structures from fungal walls that have been well-demonstrated to function as MAMPs in plants. Perception of (GlcNAc)4-8 by Arabidopsis involves CERK1, LYK4 and LYK5, three of the eight members of the LysM PRR family. We found that a glucan-enriched wall fraction from the pathogenic fungus Plectosphaerella cucumerina which was devoid of GlcNAc activated immune responses in Arabidopsis wild-type plants but not in the cerk1 mutant. Using this differential response, we identified the non-branched 1,3-ß-d-(Glc) hexasaccharide as a major fungal MAMP. Recognition of 1,3-ß-d-(Glc)6 was impaired in cerk1 but not in mutants defective in either each of the LysM PRR family members or in the PRR-co-receptor BAK1. Transcriptomic analyses of Arabidopsis plants treated with 1,3-ß-d-(Glc)6 further demonstrated that this fungal MAMP triggers the expression of immunity-associated genes. In silico docking analyses with molecular mechanics and solvation energy calculations corroborated that CERK1 can bind 1,3-ß-d-(Glc)6 at effective concentrations similar to those of (GlcNAc)4 . These data support that plants, like animals, have selected as MAMPs the linear 1,3-ß-d-glucans present in the walls of fungi and oomycetes. Our data also suggest that CERK1 functions as an immune co-receptor for linear 1,3-ß-d-glucans in a similar way to its proposed function in the recognition of fungal chito-oligosaccharides and bacterial peptidoglycan MAMPs.

Mitogen-Activated Protein Kinase Phosphatase 1 (MKP1) Negatively Regulates the Production of Reactive Oxygen Species During Arabidopsis Immune Responses.

Genetic ablation of the ß subunit of the heterotrimeric G protein complex in agb1-2 confers defective activation of microbe-associated molecular pattern (MAMP)-triggered immunity, resulting in agb1-2 enhanced susceptibility to pathogens like the fungus Plectosphaerella cucumerina BMM. A mutant screen for suppressors of agb1-2 susceptibility (sgb) to P. cucumerina BMM identified sgb10, a new null allele (mkp1-2) of the mitogen-activated protein kinase phosphatase 1 (MKP1). The enhanced susceptibility of agb1-2 to the bacterium Pseudomonas syringae pv. tomato DC3000 and the oomycete Hyaloperonospora arabidopsidis is also abrogated by mkp1-2. MKP1 negatively balances production of reactive oxygen species (ROS) triggered by MAMPs, since ROS levels are enhanced in mkp1. The expression of RBOHD, encoding a NADPH oxidase-producing ROS, is upregulated in mkp1 upon MAMP treatment or pathogen infection. Moreover, MKP1 negatively regulates RBOHD activity, because ROS levels upon MAMP treatment are increased in mkp1 plants constitutively overexpressing RBOHD (35S::RBOHD mkp1). A significant reprograming of mkp1 metabolic profile occurs with more than 170 metabolites, including antimicrobial compounds, showing differential accumulation in comparison with wild-type plants. These results suggest that MKP1 functions downstream of the heterotrimeric G protein during MAMP-triggered immunity, directly regulating the activity of RBOHD and ROS production as well as other immune responses.

Alteration of cell wall xylan acetylation triggers defense responses that counterbalance the immune deficiencies of plants impaired in the ß-subunit of the heterotrimeric G-protein.

Arabidopsis heterotrimeric G-protein complex modulates pathogen-associated molecular pattern-triggered immunity (PTI) and disease resistance responses to different types of pathogens. It also plays a role in plant cell wall integrity as mutants impaired in the Gß- (agb1-2) or Gγ-subunits have an altered wall composition compared with wild-type plants. Here we performed a mutant screen to identify suppressors of agb1-2 (sgb) that restore susceptibility to pathogens to wild-type levels. Out of the four sgb mutants (sgb10-sgb13) identified, sgb11 is a new mutant allele of ESKIMO1 (ESK1), which encodes a plant-specific polysaccharide O-acetyltransferase involved in xylan acetylation. Null alleles (sgb11/esk1-7) of ESK1 restore to wild-type levels the enhanced susceptibility of agb1-2 to the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM), but not to the bacterium Pseudomonas syringae pv. tomato DC3000 or to the oomycete Hyaloperonospora arabidopsidis. The enhanced resistance to PcBMM of the agb1-2 esk1-7 double mutant was not the result of the re-activation of deficient PTI responses in agb1-2. Alteration of cell wall xylan acetylation caused by ESK1 impairment was accompanied by an enhanced accumulation of abscisic acid, the constitutive expression of genes encoding antibiotic peptides and enzymes involved in the biosynthesis of tryptophan-derived metabolites, and the accumulation of disease resistance-related secondary metabolites and different osmolites. These esk1-mediated responses counterbalance the defective PTI and PcBMM susceptibility of agb1-2 plants, and explain the enhanced drought resistance of esk1 plants. These results suggest that a deficient PTI-mediated resistance is partially compensated by the activation of specific cell-wall-triggered immune responses.

YODA MAP3K kinase regulates plant immune responses conferring broad-spectrum disease resistance.

Mitogen-activated protein kinases (MAPKs) cascades play essential roles in plants by transducing developmental cues and environmental signals into cellular responses. Among the latter are microbe-associated molecular patterns perceived by pattern recognition receptors (PRRs), which trigger immunity. We found that YODA (YDA) - a MAPK kinase kinase regulating several Arabidopsis developmental processes, like stomatal patterning - also modulates immune responses. Resistance to pathogens is compromised in yda alleles, whereas plants expressing the constitutively active YDA (CA-YDA) protein show broad-spectrum resistance to fungi, bacteria, and oomycetes with different colonization modes. YDA functions in the same pathway as ERECTA (ER) Receptor-Like Kinase, regulating both immunity and stomatal patterning. ER-YDA-mediated immune responses act in parallel to canonical disease resistance pathways regulated by phytohormones and PRRs. CA-YDA plants exhibit altered cell-wall integrity and constitutively express defense-associated genes, including some encoding putative small secreted peptides and PRRs whose impairment resulted in enhanced susceptibility phenotypes. CA-YDA plants show strong reprogramming of their phosphoproteome, which contains protein targets distinct from described MAPKs substrates. Our results suggest that, in addition to stomata development, the ER-YDA pathway regulates an immune surveillance system conferring broad-spectrum disease resistance that is distinct from the canonical pathways mediated by described PRRs and defense hormones.

YODA Kinase Controls a Novel Immune Pathway of Tomato Conferring Enhanced Disease Resistance to the Bacterium Pseudomonas syringae.

Mitogen-activated protein kinases (MAPK) play pivotal roles in transducing developmental cues and environmental signals into cellular responses through pathways initiated by MAPK kinase kinases (MAP3K). AtYODA is a MAP3K of Arabidopsis thaliana that controls stomatal development and non-canonical immune responses. Arabidopsis plants overexpressing a constitutively active YODA protein (AtCA-YDA) show broad-spectrum disease resistance and constitutive expression of defensive genes. We tested YDA function in crops immunity by heterologously overexpressing AtCA-YDA in Solanum lycopersicum. We found that these tomato AtCA-YDA plants do not show developmental phenotypes and fitness alterations, except a reduction in stomatal index, as reported in Arabidopsis AtCA-YDA plants. Notably, AtCA-YDA tomato plants show enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and constitutive upregulation of defense-associated genes, corroborating the functionality of YDA in tomato immunity. This function was further supported by generating CRISPR/Cas9-edited tomato mutants impaired in the closest orthologs of AtYDA [Solyc08g081210 (SlYDA1) and Solyc03g025360 (SlYDA2)]. Slyda1 and Slyda2 mutants are highly susceptible to P. syringae pv. tomato DC3000 in comparison to wild-type plants but only Slyda2 shows altered stomatal index. These results indicate that tomato orthologs have specialized functions and support that YDA also regulates immune responses in tomato and may be a trait for breeding disease resistance.