PATHOGENICITY GENES IN FUNGI

Hans VanEtten, Scott Soby, Catherine Wasmann and Kevin McCluskey Plant Pathology Department University of Arizona Tucson, AZ 85721

Introduction.

This paper focuses on research reported since the 1992 ISMPMI meeting that has attempted to identify pathogenicity genes in fungi. One category we discuss consists of those genes which encode known biochemical traits that historically have been "logical" candidates for functioning in pathogenicity. In addition, we present a summary of the results of some of the newer approaches to isolate pathogenicity genes that are independent of prior knowledge of the activities encoded by the gene. In this paper we do not consider genes whose absence is required for pathogenicity (ie. avirulence genes or genes which induce a non-specific hypersensitive response).

Putative pathogenicity genes isolated because of known biochemical properties and the question of redundancy.

In the past two years a number of genes have been isolated which were anticipated to have a function in pathogenicity (Table 1). It was thought that these genes would be important because their products are enzymes that could assist a fungus in breaching the physical and chemical barriers presented by a plant, or they are part of a biosynthetic pathway that produces a phytotoxic metabolite. The roles of many of these genes in pathogenicity have been evaluated by the construction of mutants that lack a functional wild type gene through transformation-mediated gene disruption (gdr). A somewhat surprising result has been the unaltered pathogenicity of many of the gdr mutants. In several cases this might be explained by biochemical compensation due to the presence of additional genes that encode a product with the same or a similar function. For example, Cochliobolus carbonum contains three different xylanase genes so that the inactivation of one of the genes leaves two active xylanases to fulfill any putative role in pathogenicity [1, 2]. However, this explanation fails to account for the unaltered pathogenicity of gdr transformants with no detectable in vitro activity equivalent to the activity encoded by the targeted gene. For example, no residual enzymatic activity was detected in the cutinase or pisatin demethylase gdr mutants of Nectria haematococca, the cyanide hydratase (CRT) mutant of Gloeocercospora sorghi nor was there cerato-ulmin production by the gdr mutant of Ophiostoma ulmi [4, 36,45,46,47].

The most obvious interpretation of these results is that these genes have little or nothing to do with pathogenicity. For example, cutinase might be needed only for the saprophytic growth phase of N. haematococca and cerato-ulmin, a hydrophobin, might be a required

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M.l. Daniels et al. (eds.), Advances in Molecular Genetics of Plant-Microbe Interactions, Vol. 3, 163-170. 1994 Kluwer Academic Publishers.

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Table 1 . Genes cloned in the past two years as "logical" candidates for pathogenicity genes.

General Enzymatic Pathogenicity of function activity Pathogen gdr mutants l Reference

A) Cell wall and tissue degrading enzymes

Cutinases Nectria haematococca wild type [12, 36] Magnaporthe grisea wild type [39] Phytophthora capsici reduced [26]

Pectinases Penicillium olsonii wild type [18] Fusarium oxysporum

f. sp. lycospersici ? [9] Fusarium moniliforme ? [6] Sclerotinia sclerotiorum ? [7] Colletotrichum gloeosporioides ? [48]

Xylanases Cochliobolus carbonum wild type [1] Magnaporthe grisea reduced? [49]

Cellulases and other glucanases Leptosphaeria maculans ? [11]

Cochliobolus carbonum wild type [34] Uromyces viciae-/abae ? [13]

Proteases Cochliobolus carbonum ? [27] B) Phytoanticipin2

detoxifying enzymes Cyanide hydratase Gloeocercospora sorghi wild type [45,46] A venacinase Gaeumannomyces

graminis var. avenae nil [5] Tomatinase Septoria lycopersici ? [5, 33] Laccase Cryphonectria parasitica wild type [16]

C) Phytoalexin detoxifying enzymes

Pisatin demethylase Nectria haematococca reduced? [47]

Maackiain la hydroxylase Nectria haematococca ? [8]

Kievitone hydratase Fusarium solani f. sp. phaseoli ? [21] D) Toxin biosynthetic enzymes

HC toxin Cochliobolus carbonum nil [29] Siderophores Ustilago maydis wild type [24] Cerato-ulmin Ophiostoma ulmi wild type [4] Cercosporin Cercospora kikuchii nil [23] T-2 toxin Gibberella zeae reduced [14,32]

1 Nil indicates lack of pathogenicity and ? indicates effects of gdr have not yet been reported. 2Phytoanticipins are low molecular weight, antimicrobial compounds that are present in plants before challenge by microorganisms or are produced after infection solely from preexisting constituents f441.

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component of the cell wall architecture of O. ulmi [4, 36]. However, there are several other possible explanations. There may be a second set of genes with similar function that are normally produced exclusively in planta, as was demonstrated with the pectolytic enzymes of the bacterial pathogen Erwinia chrysanthemi [15]. Alternatively the assay used to assess the virulence of the gdr mutants may not be sensitive enough to measure the effect of the mutation, or the mutation may affect long-term interactions between the host and parasite, which would require extended and potentially field-based assays.

We propose another possibility, particularly for those gene products for which there is substantial but indirect evidence that they have evolved as pathogenicity traits. For example, CHT is consistently present in pathogens of cyanogenic plants, indicating that CHT may function in pathogenicity by specifically detoxifying cyanide [10, 45]. In the well studied interaction between the CHT containing G. sorghi and the cyanogenic plant sorghum: (i) cyanide serves as a fairly specific inducer of CHT and no other compound has been found that is a substrate for CHT, (ii) the product of CHT, formamide, cannot serve as a carbon or nitrogen source for G. sorghi, and (iii), the amount of CHT produced during infection is directly related to the amount of cyanogenic glucoside present in the infected tissue [10]. In addition, CHT can comprise over 4% of the total protein of this fungus after induction by cyanide [45]. It seems unlikely that such a highly expressed and specific gene would be preserved in this fungus if it served no function. A similar indication of pathogenic specialization exists for some of the other genes listed in Table 1. We propose that some of these genes represent examples of the evolution of functional redundancy for pathogenic mechanisms in phytopathogenic fungi. That is, pathogenic fungi may have evolved several means to tolerate a toxic chemical or to prevent its production, or may use both mechanical and enzymatic means to penetrate plant tissues. Therefore, no effect on pathogenicity is evident when only one of the mechanisms is eliminated. This model predicts that there will be a significant reduction in pathogenicity only when several or all redundant mechanisms are lost. Now that a number of putative pathogenicity genes have been cloned (Table 1) it should be possible to make transformants with multiple mutations to determine if redundant mechanisms can account for the apparent lack of effect when a single gene is disrupted.

Alternative approaches to identifying and evaluating putative pathogenicity genes.

In the last two years two relatively new approaches to identify pathogenicity genes in fungi have begun to gain popularity. These techniques appear to offer great potential for the identification of pathogenicity genes and require no prior knowledge of the gene's biochemical function. One approach utilizes fungal transformation vectors to both mutagenize and tag the mutated genes. The strategy is to inactivate a pathogenicity gene by integration of the vector into the gene and to detect this mutation by screening transform ants for altered pathogenicity. Increased transformation efficiency and perhaps a more dispersed distribution of integration events have been achieved with the use of the restriction enzyme-mediated integration (REMI) technique [35,38]. Insertional mutagenesis has been used to identify several genes that affect pathogenicity in Magnaporthe grisea (Table 2) and to make toxin deficient mutants of Cochliobolus heterostrophus, Cochliobolus victoriae and Phyllosticta maydis as well as non-pathogenic mutants of Alternaria alternata [38,41,52].

The other approach that is gaining popularity for identifying pathogenicity genes relies on the induction of fungal transcripts under conditions in which one would expect pathogenicity genes to be expressed. Induced transcripts are detected by a differential screen, followed by a functional assay in a transformation-mediated gdr mutant (Table 2). Fungal

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Table 2. Alternative approaches to identify pathogenicity genes.

Gene or gene Pathogenicity of Technique Pathogen product identified gdr mutant Reference

A) Insertional mutagenesis with or without REMI

I) Magnaporthe grisea a regulator of glucose repression? reduced [38]

an acyltransferase reduced [38] a cAMP-dependent

protein kinase reduced [38] B) In planta produced

fungal transcripts 1) Phytophthora infestans ubiquitin, ? [30]

calmodulin ? [30] glycine-rich protein

(cell wall protein?) ? [30] cell adhesion protein? ? [30]

2) Magnaporthe grisea a hydrophobin reduced [40] C) In planta-produced fungal proteins

1) Cladosporium fulvum two extracellular proteins wild type [19, 42]

D) Starvation induced transcripts 1) Cladosporium fulvum alcohol dehydrogenase ? [28]

aldehyde dehydrogenase ? [28] E) Stage specific transcripts

1) Haploid to dikaryon infectious stage, Ustilago maydis cellulase wild type [3]

2) Infection structure fonnation Uromyces appendiculatus

a cytoskeleton protein? ? [50, 51] a cell adhesion protein? ? [50,51]

Magnaporthe grisea mif23 and mif29 ? [20]

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growth in planta, under nutrient deprivation, or during specific developmental stages associated with the pathogenic growth stage have been sources of transcripts. A variation of this general strategy is to identify fungal proteins produced in planta and then to identify putative pathogenicity genes by reverse genetics.

The power of both of these approaches is evident by the number of genes that have been identified which were not previously "logical" candidates for pathogenicity genes (Table 2). One future challenge will be to distinguish between genes which have pleiotropic effects on pathogenicity because their functions are essential for normal cell growth (eg. calmodulin) from those which have evolved specifically for pathogenicity on plants. We speculate that one class of pathogenicity genes that will be identified by these approaches will consist of gene products that suppress active resistance mechanisms in plants [37].

Dispensable chromosomes in phytopathogenic fungi as a source of pathogenicity genes.

Previous work [25] has shown that genes (PDA) for detoxifying the pea phytoalexin pisatin can be on dispensable (DS) chromosomes in N. haematococca. Recently a gene (MAK1) for detoxifying chickpea phytoalexins (medicarpin and maackiain) has been isolated from a DS chromosome of N. haematococca [8]. Although PDA and MAK are putative pathogenicity genes in this organism, gdr mutants of PDA retain virulence on pea [47]. However, if the DS chromosome containing PDA is lost, the isolates become non-pathogenic, implying that other genes needed for pathogenicity on pea are also located on the DS chromosome [43].

The location of pathogenicity genes on a defined and dispensable portion of the genome not only has significant evolutionary implications for the origin of pathogenicity genes but also enhances our ability to identify these genes. Variations on the approaches illustrated in Table 2 to identify transcripts of pathogenicity genes can be used with the added advantage that the DS chromosomes themselves can be used as probes to detect DS chromosome-derived transcripts. Furthermore, since these chromosomes are not essential for growth in culture, manipulations that cause partial deletions of the chromosome, combined with a virulence assay can be used to map the locations of these pathogenicity genes. An elegant method that causes directed partial chromosomal deletions in N. haematococca has been developed by Kistler and co-workers [17]. By constructing transformation vectors with telomeric sequences, to cause chromosome breakage, and DS chromosomal DNA to target the integration event, they have been able to produce non-pathogenic transformants containing truncated DS chromosomes. This should allow the identification of pea pathogenicity genes located on the deleted portion of the chromosome by transformation of the non-pathogenic transformants with cloned DNA from this portion of the chromosome.

Pathogenicity genes on DS chromosomes or dispensable portions of chromosomes have also been observed in C. carbonum and there are preliminary indications that Colletotrichum gloeosporioides may have pathogenicity genes on DS chromosomes [22,29, 31]. Therefore it may not be unusual for pathogenicity genes to be located on DS chromosomes. This will not only facilitate the isolation and identification of pathogenicity genes but could have significant implications for how pathogenicity has evolved and is maintained in fungi.

Acknowledgements.

We wish to thank all those individuals who shared unpublished information with us. Others in addition to those cited have proposed similar ideas or have preliminary indications of the cloning of similar genes but we limited our discussion to those in which we had knowledge

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that functional genes had been cloned or disrupted. We apologize for any oversights we may have made.

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