Expression polymorphism at the ARPC4 locus links the actin cytoskeleton with quantitative disease resistance to Sclerotinia sclerotiorum in Arabidopsis thaliana.

Quantitative disease resistance (QDR) is a form of plant immunity widespread in nature, and the only one active against broad host range fungal pathogens. The genetic determinants of QDR are complex and largely unknown, and are thought to rely partly on genes controlling plant morphology and development. We used genome-wide association mapping in Arabidopsis thaliana to identify ARPC4 as associated with QDR against the necrotrophic fungal pathogen Sclerotinia sclerotiorum. Mutants impaired in ARPC4 showed enhanced susceptibility to S. sclerotiorum, defects in the structure of the actin filaments and in their responsiveness to S. sclerotiorum. Disruption of ARPC4 also alters callose deposition and the expression of defense-related genes upon S. sclerotiorum infection. Analysis of ARPC4 diversity in A. thaliana identified one haplotype (ARPC4R ) showing a c. 1 kbp insertion in ARPC4 regulatory region and associated with higher level of QDR. Accessions from the ARPC4R haplotype showed enhanced ARPC4 expression upon S. sclerotiorum challenge, indicating that polymorphisms in ARPC4 regulatory region are associated with enhanced QDR. This work identifies a novel actor of plant QDR against a fungal pathogen and provides a prime example of genetic mechanisms leading to the recruitment of cell morphology processes in plant immunity.

Parallel evolution of the POQR prolyl oligo peptidase gene conferring plant quantitative disease resistance.

Plant pathogens with a broad host range are able to infect plant lineages that diverged over 100 million years ago. They exert similar and recurring constraints on the evolution of unrelated plant populations. Plants generally respond with quantitative disease resistance (QDR), a form of immunity relying on complex genetic determinants. In most cases, the molecular determinants of QDR and how they evolve is unknown. Here we identify in Arabidopsis thaliana a gene mediating QDR against Sclerotinia sclerotiorum, agent of the white mold disease, and provide evidence of its convergent evolution in multiple plant species. Using genome wide association mapping in A. thaliana, we associated the gene encoding the POQR prolyl-oligopeptidase with QDR against S. sclerotiorum. Loss of this gene compromised QDR against S. sclerotiorum but not against a bacterial pathogen. Natural diversity analysis associated POQR sequence with QDR. Remarkably, the same amino acid changes occurred after independent duplications of POQR in ancestors of multiple plant species, including A. thaliana and tomato. Genome-scale expression analyses revealed that parallel divergence in gene expression upon S. sclerotiorum infection is a frequent pattern in genes, such as POQR, that duplicated both in A. thaliana and tomato. Our study identifies a previously uncharacterized gene mediating QDR against S. sclerotiorum. It shows that some QDR determinants are conserved in distantly related plants and have emerged through the repeated use of similar genetic polymorphisms at different evolutionary time scales.

Probing differentially expressed genes against a microarray database for in silico suppressor/enhancer and inhibitor/activator screens.

High-density oligonucleotide arrays are widely used for analysis of gene expression on a genomic scale, but the generated data remain largely inaccessible for comparative analysis purposes. Similarity searches in databases with differentially expressed gene (DEG) lists may be used to assign potential functions to new genes and to identify potential chemical inhibitors/activators and genetic suppressors/enhancers. Although this is a very promising concept, it requires the compatibility and validity of the DEG lists to be significantly improved. Using Arabidopsis and human datasets, we have developed guidelines for the performance of similarity searches against databases that collect microarray data. We found that, in comparison with many other methods, a rank-product analysis achieves a higher degree of inter- and intra-laboratory consistency of DEG lists, and is advantageous for assessing similarities and differences between them. To support this concept, we developed a tool called MASTA (microarray overlap search tool and analysis), and re-analyzed over 600 Arabidopsis microarray expression datasets. This revealed that large-scale searches produce reliable intersections between DEG lists that prove to be useful for genetic analysis, thus aiding in the characterization of cellular and molecular mechanisms. We show that this approach can be used to discover unexpected connections and to illuminate unanticipated interactions between individual genes.

Dissection of the complex phenotype in cuticular mutants of Arabidopsis reveals a role of SERRATE as a mediator.

Mutations in LACERATA (LCR), FIDDLEHEAD (FDH), and BODYGUARD (BDG) cause a complex developmental syndrome that is consistent with an important role for these Arabidopsis genes in cuticle biogenesis. The genesis of their pleiotropic phenotypes is, however, poorly understood. We provide evidence that neither distorted depositions of cutin, nor deficiencies in the chemical composition of cuticular lipids, account for these features, instead suggesting that the mutants alleviate the functional disorder of the cuticle by reinforcing their defenses. To better understand how plants adapt to these mutations, we performed a genome-wide gene expression analysis. We found that apparent compensatory transcriptional responses in these mutants involve the induction of wax, cutin, cell wall, and defense genes. To gain greater insight into the mechanism by which cuticular mutations trigger this response in the plants, we performed an overlap meta-analysis, which is termed MASTA (MicroArray overlap Search Tool and Analysis), of differentially expressed genes. This suggested that different cell integrity pathways are recruited in cesA cellulose synthase and cuticular mutants. Using MASTA for an in silico suppressor/enhancer screen, we identified SERRATE (SE), which encodes a protein of RNA-processing multi-protein complexes, as a likely enhancer. In confirmation of this notion, the se lcr and se bdg double mutants eradicate severe leaf deformations as well as the organ fusions that are typical of lcr and bdg and other cuticular mutants. Also, lcr does not confer resistance to Botrytis cinerea in a se mutant background. We propose that there is a role for SERRATE-mediated RNA signaling in the cuticle integrity pathway.

Misexpression of FATTY ACID ELONGATION1 in the Arabidopsis epidermis induces cell death and suggests a critical role for phospholipase A2 in this process.

Very-long-chain fatty acids (VLCFAs) are important functional components of various lipid classes, including cuticular lipids in the higher plant epidermis and lipid-derived second messengers. Here, we report the characterization of transgenic Arabidopsis thaliana plants that epidermally express FATTY ACID ELONGATION1 (FAE1), the seed-specific beta-ketoacyl-CoA synthase (KCS) catalyzing the first rate-limiting step in VLCFA biosynthesis. Misexpression of FAE1 changes the VLCFAs in different classes of lipids but surprisingly does not complement the KCS fiddlehead mutant. FAE1 misexpression plants are similar to the wild type but display an essentially glabrous phenotype, owing to the selective death of trichome cells. This cell death is accompanied by membrane damage, generation of reactive oxygen species, and callose deposition. We found that nuclei of arrested trichome cells in FAE1 misexpression plants cell-autonomously accumulate high levels of DNA damage, including double-strand breaks characteristic of lipoapoptosis. A chemical genetic screen revealed that inhibitors of KCS and phospholipase A2 (PLA2), but not inhibitors of de novo ceramide biosynthesis, rescue trichome cells from death. These results support the functional role of acyl chain length of fatty acids and PLA2 as determinants for programmed cell death, likely involving the exchange of VLCFAs between phospholipids and the acyl-CoA pool.

The epidermis-specific extracellular BODYGUARD controls cuticle development and morphogenesis in Arabidopsis.

The outermost epidermal cell wall is specialized to withstand pathogens and natural stresses, and lipid-based cuticular polymers are the major barrier against incursions. The Arabidopsis thaliana mutant bodyguard (bdg), which exhibits defects characteristic of the loss of cuticle structure not attributable to a lack of typical cutin monomers, unexpectedly accumulates significantly more cell wall-bound lipids and epicuticular waxes than wild-type plants. Pleiotropic effects of the bdg mutation on growth, viability, and cell differentiation are also observed. BDG encodes a member of the alpha/beta-hydrolase fold protein superfamily and is expressed exclusively in epidermal cells. Using Strep-tag epitope-tagged BDG for mutant complementation and immunolocalization, we show that BDG is a polarly localized protein that accumulates in the outermost cell wall in the epidermis. With regard to the appearance and structure of the cuticle, the phenotype conferred by bdg is reminiscent of that of transgenic Arabidopsis plants that express an extracellular fungal cutinase, suggesting that bdg may be incapable of completing the polymerization of carboxylic esters in the cuticular layer of the cell wall or the cuticle proper. We propose that BDG codes for an extracellular synthase responsible for the formation of cuticle. The alternative hypothesis proposes that BDG controls the proliferation/differentiation status of the epidermis via an unknown mechanism.