Pectin degradation by Botrytis cinerea: recognition of endopolygalacturonases by an Arabidopsis receptor and utilization of Dgalacturonic acid

The necrotrophic fungal plant pathogen<em>Botrytis cinerea</em> is able to infect over 200 host plants and cause severe damage to crops, both pre- and post-harvest. <em>B. cinerea</em> often penetrates host leaf tissue at the anticlinal cell wall and subsequently grows into and through the middle lamella, which consists mostly of low-methylesterified pectin. Effective pectin degradation thus is important for virulence of <em>B. cinerea</em>. <strong>Chapter 1</strong> describes the chemical structures of plant cell wall polysaccharides, the cell wall-associated mechanisms that confer resistance against pathogens, and the microbial enzymes involved in cell wall decomposition. It then discusses the plant cell wall degrading enzymes of pathogenic fungi and illustrates with case studies the process of pectin decomposition by <em>B. cinerea</em>. <strong>Chapter 2</strong>describes the molecular identification and functional characterization of a novel MAMP receptor RBPG1, a Leucine-Rich Repeat Receptor-Like Protein (LRR-RLP), that recognizes fungal endo-polygalacturonases (endo-PGs), in particular the <em>B. cinerea </em>protein BcPG3. Infiltration of the BcPG3 protein into <em>Arabidopsis thaliana </em>accession Col-0 induced a necrotic response. Heat-inactivated protein and a catalytically inactive mutant protein retained the ability to induce necrosis. An 11-amino acid peptide stretch was identified that is conserved among many fungal but not plant endo-PGs. A synthetic peptide comprising this sequence was unable to induce necrosis. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the <em>RBPG1</em> gene, which is required for responsiveness of <em>A. thaliana</em> to the BcPG3 protein. Co-immunoprecipitation experiments demonstrated that RBPG1 and BcPG3 form a complex in<em>Nicotiana benthamiana, </em>which also involves the <em>A. thaliana </em>LRR-RLK SOBIR1. The <em>sobir1</em> mutant plants no longer respond to BcPG3. Furthermore, overexpression of RBPG1 in the BcPG3-non-responsive accession Br-0 did not enhance resistance to a number of microbial pathogens. <strong>Chapter 3</strong>describes the functional, biochemical and genetic characterization of the D-galacturonic acid catabolic pathway in <em>B. cinerea</em>. The <em>B. cinerea</em> genome contains two non-homologous galacturonate reductase genes (<em>Bcgar</em>1 and <em>Bcgar</em>2), a galactonate dehydratase gene (<em>Bc</em><em>lgd</em>1), and a 2-keto-3-deoxy-L-galactonate aldolase gene (<em>Bc</em><em>l</em><em>ga</em>1). Targeted gene replacement of all four genes in <em>B. cinerea</em>, either separately or in combinations, yielded mutants that were affected in growth on D-galacturonic acid, pectate, or pectin as the sole carbon source. The extent of growth reduction of the mutants on pectic substrates was positively correlated to the proportion of D-galacturonic acid present in the pectic substrate. The virulence of these mutants on different host plants is discussed in <strong>Chapter 4</strong>. These mutants showed reduced virulence on <em>N. benthamiana</em> and <em>A. thaliana </em>leaves, but not on tomato leaves. The cell walls of <em>N. benthamiana</em> and <em>A. thaliana</em> leaves have a higher D-galacturonic acid content as compared to tomato. Additional <em>in vitro</em> growth assays with the knockout mutants suggested that the reduced virulence of D-galacturonic acid catabolism-deficient mutants on <em>N. benthamiana</em> and <em>A. thaliana</em> is not only due to the inability of the mutants to utilize an abundant carbon source as nutrient, but also due to the growth inhibition by catabolic intermediates. In <strong>Chapter 5</strong>, the functional characterization of two putative D-galacturonic acid transporters is presented. <em>Bchxt</em>15 is highly and specifically induced by D-galacturonic acid, whereas <em>Bchxt</em>13 is highly expressed in the presence of all carbon sources tested except for glucose. Subcellular localization of BcHXT13-GFP and BcHXT15-GFP fusion proteins expressed under their native promoter suggests that the fusion proteins are localized in plasma membranes and intracellular vesicles. Knockout mutants in the <em>Bchxt</em>13 and <em>Bchxt</em>15 genes, respectively, were neither affected in their growth on D-galacturonic acid as the sole carbon source, nor in their virulence on tomato and <em>N. benthamiana</em> leaves. <strong>Chapter 6</strong>describes the genome-wide transcriptome analysis in <em>B. cinerea </em>grown in media containing glucose and pectate as sole carbon sources. Genes were identified that are significantly altered in their expression during growth on these two carbon sources. Conserved sequence motifs were identified in the promoters of genes involved in pectate decomposition and D-galacturonic acid utilization. The role of these motifs in regulating D-galacturonic acid-induced expression was functionally analysed in thepromoter of the <em>Bclga</em>1 gene, which encodes one of the key enzymes in the D-galacturonic acid catabolic pathway. The regulation by D-galacturonic acid required the presence of sequences encompassing the GAE1 motif and a binding motif for the pH-dependent transcriptional regulator PacC. <strong>Chapter 7 </strong>provides a general discussion of the results presented in this thesis. A model of the concerted action of pectin degradation and subsequent monosaccharide consumption and co-regulation of gene expression is proposed.