Methods paperEvolutionary analysis of glycosyl hydrolase family 28 (GH28) suggests lineage-specific expansions in necrotrophic fungal pathogens
Introduction
Gene families are sets of genes that have arisen through the accumulation of duplicate copies of a single ancestral gene. Following duplication, the paralogous genes represent a genetic redundancy that may result in relaxation of selection pressure on one or both of the gene copies, allowing mutations to accumulate in one or both copies (Ohno, 1970, Prince and Pickett, 2002). Gene copies that incorporate deleterious mutations are often purged from the genome via purifying selection, while copies that acquire novel or enhanced functionality can become fixed in the population through positive or diversifying selection (Hughes, 2002, Lynch and Conery, 2003). This pattern of duplication and expansion of advantageous gene copies paired with the expulsion of dysfunctional gene copies has been described as evolution via the birth-and-death process (Nei et al., 1997, Nei and Rooney, 2005).
Glycosyl hydrolase family 28 (GH28) has interesting functional diversity and is variable in copy number among related organisms, making this gene family a likely candidate for birth-and-death evolution. GH28 enzymes are involved in degradation of pectin, a major structural constituent of the plant cell wall. Pectin is a long polysaccharide chain that is composed of α-linked galacturonic acid (GalA) monomers, with some regions of GalA alternating with rhamnose or branched xylose side-chains (Willats et al., 2006). Various enzymes in family GH28 degrade these bonds by catalyzing a series of functionally distinct reactions. Endo-glycosidases catalyze hydrolysis of internal glycosidic bonds at random locations within the polysaccharide, while exo-glycosidases catalyze hydrolysis of terminal bonds that attach individual sugars to the ends of the polysaccharides. GH28 enzymes are also categorized into polygalacturonases (PG), which hydrolyze GalA-GalA linkages (E.C.'s 3.2.1.15 [endo-PG] and 3.2.1.67 [exo-PG]), rhamnogalacturonases (RG), which hydrolyze GalA-rhamnose bonds (E.C. 3.2.1.-), and xylogalacturonases (XG), which hydrolyze GalA-xylose bonds (E.C. 3.2.1.-) (Markovič and Janeček, 2001).
Genes encoding GH28 enzymes have been identified in genomes of a wide range of plants, bacteria, and fungi. Fungal GH28 pectinases are degradative enzymes used by both saprotrophs growing on senesced leaf tissue (Kjøller and Struwe, 2002) and plant pathogens seeking to gain access to plant intracellular nutrients (Herron et al., 2000, Reignault et al., 2008). The contribution of pectinases to pathogenic infectivity remains unclear. Targeted gene disruption studies have demonstrated that various GH28 functional types do play a role in virulence (Garcia-Maceira et al., 2001, Kars et al., 2005, Shieh et al., 1997, ten Have et al., 1998), while several other studies have not found GH28 expression to be critically important during cell wall invasion (Scott-Craig et al., 1990, Gao et al., 1996). This disagreement may be due to lack of consideration of the differing strategies adopted by phytopathogenic fungi (Glazebrook, 2005). Necrotrophic pathogens acquire carbon and energy by extensively degrading plant tissue, often resulting in the host's death. In contrast, biotrophic fungi obtain nutrients from living plant tissues and depend on continued survival of their host (Oliver and Ipcho, 2004). Therefore, while necrotrophs indiscriminately destroy cell wall constituents, biotrophs colonize intact plant cells while avoiding immune responses. Extracellular enzymes like GH28s can elicit a wide array of plant immunological responses, such as the production of extracellular plant proteins that inhibit fungal PGs, known as polygalacturonase-inhibiting proteins or PGIPs (De Lorenzo et al., 2001, Federici et al., 2001). GH28 gene products also stimulate the PGIP-mediated hypersensitive immune response, or localized cell death, in host plants. Both PGs and PGIPs are highly polymorphic, resulting in a high level of PGIP specificity for particular PG isozymes (Casasoli et al., 2009, Cook et al., 1999, De Lorenzo et al., 2001, Di Matteo et al., 2006, Raiola et al., 2008). This molecular co-evolutionary arms race could be a source of strong diversifying selection on the GH28 repertoires of fungal necrotrophs. Conversely, plant immune system activity likely acts to reduce GH28 diversity in biotrophic fungi via purifying selection (Oliver and Ipcho, 2004). Furthermore, saprotrophic fungi that degrade dead plant material would not experience the strong diversifying selection associated with a co-evolutionary arms race, and should therefore also possess a low level of GH28 diversity.
Because of the importance of pectinase in liberating carbon and energy and the wide distribution of PGIPs, we hypothesize that the distribution of GH28 genes in fungi may be closely linked to the evolution of ecological strategy and pathogenic niche. In this study, we examine the pattern of GH28 gene family evolution by investigating its occurrence and distribution in fungal genomes and by comprehensively reconstructing the long-term evolutionary history of the GH28 family in fungi. Using GH28 genes from completely sequenced fungal genomes, we infer the ancestral gene copy number in most recent common ancestors (MRCA) of major taxonomic groups. Additionally, using a reconstructed phylogeny of fungal GH28 sequences, we infer the ancestral enzymatic mode(s) of action and subsequent functional diversification within this gene family. We then test the hypothesis that the occurrence of GH28s, or particular functional categories of GH28, is correlated with ecological strategy (pathogenic vs. non-pathogenic) and pathogenic niche (necrotrophic vs. biotrophic) against the null hypothesis that GH28 copy number is simply the result of variation in overall genome size.
Section snippets
Fungal SSU rRNA-based species tree and ecological characteristics
We first reconstructed a species tree using small subunit ribosomal RNA (SSU rRNA) sequences from 69 fully sequenced fungal genomes (Supplementary File 1), including 57 Ascomycetes, 8 Basidiomycetes, 3 Mucoromycotinas, and 1 Chytridiomycota, as well as an additional 2 Oomycetes (Stramenopiles) and 1 plant. Relevant ribosomal sequences were extracted from the SILVA database (Pruesse et al., 2007) and GenBank (Benson et al., 2005) and aligned using the MUSCLE sequence alignment tool using default
GH28 distribution and ancestral copy number
The survey of GH28 members revealed the presence of at least one GH28 homolog in 40 of the 69 completed fungal genome sequences examined, with GH28 copy number per genome ranging from 0 to 20 (Supplementary File 1). Such large variation is suggestive of a dynamic evolutionary history, and is consistent with evolution via the birth-and-death process, resulting in lineage-specific gene family expansions and contractions (Nei et al., 1997, Nei and Rooney, 2005).
We used a maximum parsimony approach
Conclusions
In this study, we comprehensively surveyed GH28 gene family distribution and diversity within the fungal kingdom, and characterized its evolution as being consistent with the birth-and-death model. The initial appearance of GH28s predates the evolution of fungi, which places the origin of this gene family at more than 1.5 billion years ago. Ancient gene families such as GH28 continue to radiate and expand into new molecular niches. Our analysis indicates that the earliest fungi already
Acknowledgments
The authors thank Bess Heidenreich for assistance in compilation of data. Financial support for the work was provided by Kent State University.
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