Reconstructing the diversification of subtilisins in the pathogenic fungus Metarhizium anisopliae
Introduction
Host–pathogen interactions are an important force shaping organismal diversity, yet little is known about the evolution of genes responsible for resistance in the host or virulence in the pathogen (Stotz et al., 2000). This is particularly true for fungal pathogens, even though they are the principal disease causing agents of plants, and of many animals including insects (St. Leger and Screen, 2001). Efforts to understand the molecular parameters of fungal virulence have largely focused on the complex array of enzymes they secrete during penetration and colonization as they are thought to be primarily involved in this necrotrophic stage of pathogenesis (Walton, 1996). It is inferred that the production of enzymes that disrupt the physiological integrity of hosts will have a strong selective advantage for pathogens. Most, if not all, fungal genes encoding hydrolytic enzymes are members of gene families—genes of common origin that encode products of similar function (Walton, 1996). In theory, after gene duplication, one gene copy maintains the original function, while the other copy is free to accumulate amino-acid changes toward functional divergence. Consequently, gene duplication and divergence play an important role in generating the functional diversification necessary for adaptation (Graur and Li, 2000). Presumably, this process produced the complex array of chemical weaponry that enables ascomycete fungi to adapt to a broad array of habitats as saprophytes and pathogens. However, the means by which gene families develop and diversify in function is not well understood in any clan of organisms. In particular, compared to genes fundamental to morphological and developmental features, the contribution gene families make to ecological diversification and the nature of the evolutionary forces acting during this process are very poorly known. In part, this is because genes directly involved in ecological attributes are hard to identify (Duda and Palumbi, 1999).
Pathogenic fungi, such as the insect pathogen Metarhizium anisopliae, provide an unusual model system to help remedy this deficiency as the enzymes they secrete into the host environment can prima facie be defined as ecological traits. Thus, by comparing gene divergence and expression in genetically and ecologically distinct strains, it should be possible to determine the role of genes in ecological diversification and the nature of the evolutionary forces acting during this process. Subtilisins are ubiquitous fungal proteases that provide an excellent model for this work. Site-directed mutagenesis and protein engineering have provided intimate knowledge of structure–function relationships in subtilisins. It is possible by comparing subtilisins to distinguish from a myriad of possible amino-acid replacements a large number already known to cause functional differences. Such an approach can unite form, function and phylogeny to glean insight into the process of molecular adaptation (Golding and Dean, 1998). In the case of M. anisopliae, subtilisins are intricately related to a strains abilities to penetrate, colonize and macerate insect host tissues (St. Leger et al., 1996), and they are under evolutionary pressure to respond to hosts which may themselves undergo relatively rapid changes in levels and types of protease inhibitor that can provide a barrier to infection (Boucias and Pendland, 1987). Subtilisins therefore provide an opportunity in a fungus to study a multigene family that may be duplicating and diversifying while under strong selective pressure.
In the current study, we investigated whether subtilisins have diversified in M. anisopliae, how subtilisins are evolving among strains and whether subtilisin diversification is generally maintained between loci, within loci or both. To perform this work, we obtained three related data sets. First, we acquired comparative sequence data from three well-characterized strains of M. anisopliae. Two of these strains have multiple hosts. The third strain is specific against acridids (locusts) allowing us to investigate whether adaptation to a specific host has altered evolutionary trends. Second, a phylogeny was established to demonstrate how the analyzed sequences are evolutionarily related. Finally, the results obtained by sequence comparison were interpreted by homology modeling based on well-characterized subtilisins.
Section snippets
Organisms
We employed three strains from the U.S. Department of Agriculture Entompathogenic Fungus Collection in Ithaca, NY. M. anisopliae sf. anisopliae strains 820 (origin: France—coleopteran host) and 2575 (Origin: N. America—coleopteran host, a multi-host strain in laboratory conditions) and M. anisopliae sf. acridium 324 (Australian—specific for acridids). These are in different genotypic classes as judged by allozyme analysis (St. Leger et al., 1992). Cultures were maintained on potato dextrose
Nucleotide and amino acid sequences for M. anisopliae strain 2575
All 11 genes from strain 2575 were originally detected in an EST database, i.e., in cDNA. This indicates that they are all expressed and presumably functional. Restriction digests of genomic DNA from 2575 were probed with cDNA from each of the subtilisin genes and in each case revealed only single bands consistent with their being single copy genes (not shown).
The calculated molecular weights of the subtilisin proteases range from 35.1 to 57 kDa. The preregion, demonstrated by Von Heijne (1986)
Discussion
The evolution of subtilisins has resulted in a relatively large number of isoforms in the pathogenic fungus M. anisopliae and in fewer isoforms in saprophytic and plant pathogenic fungi (Siezen and Leunissen, 1997). The insect pathogen Verticillium lecanii expresses more types and higher levels of subtilisins than does the plant pathogen Verticillium albo-atrum (St. Leger et al., 1997), so the divergence of extra subtilisins may have occurred independently in different lineages of pyrenomycete
Acknowledgements
This work was supported by the NSF (grant MCB-0091196) and the USDA (grant 2001-35302-10141). We would like to thank A. Wilson for computer support.
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