The role of pectin degradation in pathogenesis of Botrytis cinerea Ilona Kars Promotor: Prof. Dr. Ir. P.J.G.M. de Wit Hoogleraar Fytopathologie Wageningen Universiteit Co-promotor: Dr. J.A.L. van Kan Universitair docent, Laboratorium voor Fytopathologie Wageningen Universiteit Promotiecommissie: Prof. Dr. Ir. A.G.J. Voragen, Wageningen Universiteit Prof. Dr. Ir. C.M.J. Pieterse, Universiteit Utrecht Dr. Ir. G. Smant, Wageningen Universiteit Dr. M. Rep, Universiteit van Amsterdam Dit onderzoek is uitgevoerd binnen de onderzoeksschool Experimental Plant Sciences. The role of pectin degradation in pathogenesis of Botrytis cinerea Ilona Kars Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. Dr. M.J. Kropff, in het openbaar te verdedigen op vrijdag 21 september 2007 des morgens te 11.00 uur in de Aula. Title The role of pectin degradation in pathogenesis of Botrytis cinerea Author Ilona Kars Thesis Wageningen University, Wageningen, NL (2007) with references, with summaries in Dutch and English ISBN 978-90-8504-707-0 Table of contents Chapter 1 General introduction 7 Chapter 2 Functional analysis of Botrytis cinerea pectin methylesterase genes by PCR-based targeted mutagenesis: Bcpme1 and Bcpme2 are dispensable for virulence of strain B05.10 25 Chapter 3 Necrotizing activity of five Botrytis cinerea endopolygalacturonases produced in Pichia pastoris 47 Chapter 4 Functional analysis of Botrytis cinerea endopolygalacturonases Bcpg3-6 69 Chapter 5 A polygalacturonase inhibiting protein from grapevine reduces the symptoms of the endopolygalacturonase BcPG2 from Botrytis cinerea in Nicotiana benthamiana leaves without any evidence for in vitro interaction 83 Chapter 6 The quantitative trait locus RBPG1 controls the response to Botrytis cinerea polygalacturonases in Arabidopsis thaliana 105 Chapter 7 General discussion 123 References 133 Summary / Samenvatting 153 Dankwoord 159 About the author / Over de auteur 163 Publications 165 Education statement graduateschool Experimental Plant Sciences 167 Chapter 1 General introduction Ilona Kars and Jan A.L. van Kan This chapter is published as: Kars, I., and van Kan, J.A.L. (2004) Extracellular enzymes and metabolites involved in pathogenesis of Botrytis. In Botrytis: Biology, Pathology and Control (Elad, Y., Williamson, B., Tudzynski, P. and Nelen, N., eds). Dordrecht: Kluwer Academic Publishers, pp. 99-118. Chapter 1 Abstract The infection of host plants by Botrytis spp. is mediated by numerous extracellular enzymes and metabolites. Each of these compounds may play a role in different stages of the infection process. Cutinases, lipases and some cell wall-degrading enzymes may facilitate the penetration of the host surface, while toxins, oxalate and reactive oxygen species may contribute to killing of the host cells. Several cell wall-degrading enzymes contribute to the conversion of host tissue into fungal biomass, but also other enzymes, such as laccases and proteases are potentially involved in pathogenesis. The cloning of the corresponding genes in recent years has facilitated studies on gene expression and targeted mutagenesis. This chapter gives an updated overview of the research performed on these secreted enzymes and metabolites and the role they play in pathogenesis. 8 General introduction Introduction Botrytis cinerea is able to infect a wide spectrum of host plant species, whereas other Botrytis species are confined to a single host species. All Botrytis species, whether specific or not, are necrotrophs implying they are able to kill host cells during the infection process. De Bary (1886) observed that carrot cells were killed in advance of invading hyphae of the soft rot fungus Sclerotinia. He also noted that fluid from rotten tissue could degrade healthy host tissue, while boiled fluid had no effect. This led to his conclusion that the fungus produced heat-labile enzymes and toxins that kill and degrade plant cells. The same is true for Botrytis species. They are equipped with a set of enzymes and/or metabolites that enable the pathogen to invade host tissue, kill host cells and eventually convert host tissue into fungal biomass. Many of these enzymes and metabolites act extracellularly at the plant- fungus interface, or even in the host tissue at some distance from the growing hyphae. This chapter will deal with pathogenicity factors, i.e. effector molecules that cause damage to the host thereby enabling the pathogen to complete its disease and life cycle. The emphasis is placed on fungal extracellular enzymes, but we will also discuss the biosynthetic pathways of metabolites secreted during pathogenesis. This chapter discusses neither differentiation of infection structures nor the production of phytohormones. Most data originate from the research on B. cinerea, but we will also discuss other Botrytis species where relevant information is available. Penetration of the host surface The disease cycle starts with a conidium landing on the host surface. Upon attachment, it germinates on the host surface and produces a germtube that develops into an appressorium that facilitates penetration of the host surface. Invasion of host tissue can be achieved by active penetration or passive ingress. B. cinerea is an opportunist that can initiate infection at wound sites, or at sites previously infected by other pathogens. Nevertheless, Botrytis spp. are perfectly able to penetrate intact host surfaces. Only direct, active penetration of the epidermal surface is discussed in this section. For reasons of simplicity the penetration of dead or wounded tissue is regarded as an expansion process rather than a penetration process and is dealt with in later sections. The first barrier to breach is the host cuticle covering all aerial parts of the plant. The cuticle consists of cutin, a polyester of hydroxylated and epoxidised C16- and C18-fatty acids, in many cases covered with a hydrophobic wax layer consisting of fatty alcohols. Physical damage or brute mechanical penetration of the cuticle by B. cinerea is not usually observed (Cole et al., 1996; Williamson et al., 1995) indicating that enzymatic activity is involved in penetrating intact host surfaces (Salinas and Verhoeff, 1995). 9 Chapter 1 The role of lipase in wax layer penetration and surface adhesion The wax layer does not seem to pose a serious barrier, although removal of the wax layer by abrasion was reported to increase the infection incidence (Sutton et al., 1984). No correlation was observed between the wax layer dry weight of rose or gerbera petals and their susceptibility to B. cinerea (Kerssies and Frinking, 1996). It is conceivable that B. cinerea produces surfactants: proteins or metabolites that reduce surface hydrophobicity and “dissolve” the wax layer, thereby providing access to the underlying cutin polymer. The polysaccharide cinerean, covering B. cinerea germ tubes might fulfill a role as surfactant. Alternatively, the reduction of host surface tension may be achieved enzymatically. Cutinases, serine esterases, lipases and other non-specified esterases are reported to be involved in the adhesion of several plant pathogenic fungi: Alternaria brassicola (Berto et al., 1997; Fan and Köller, 1998; Köller et al., 1995; Yao and Köller, 1995), Colletotrichum graminicola (Pascholati et al., 1993), Erysiphe graminis (Pascholati et al., 1992), Uromyces viciae-fabae (Deising et al., 1992; Clement et al., 1993a, b). B. cinerea produces an extracellular triacylglycerol lipase, with a molecular mass of 60 kDa during culture in the presence of a fatty acid ester. The enzyme is able to hydrolyse unsaturated long chain fatty acid esters (Comménil et al., 1995), known to be components of cutin and waxes. Lipase production in vitro was induced by wax esters and free fatty acids (Comménil et al., 1999). The lipase possesses cutinolytic activity, although its kinetic properties (Comménil et al., 1998) are clearly distinct from those of a 'typical' cutinase that will be discussed below. It was proposed that the enzyme plays a role in modifying the waxes and cuticle, and in adhesion of conidia to the plant surface (Comménil et al., 1997, 1998). Studies with polyclonal antibodies blocking the active site suggested that the lipase plays an important role in the infection process. When antibodies were applied on to intact tomato leaves prior to inoculation with B. cinerea conidia, the fungal germ tubes were unable to penetrate the cuticle (Comménil et al., 1998). The antibodies did not affect germination of B. cinerea conidia nor did they inhibit the infection of wounded tissue, suggesting a role for the lipase specifically during host surface penetration. These antibodies also inhibited a lipase purified from Alternaria brassicicola and they were able to reduce by 90% the occurrence on intact cauliflower leaves of blackspot lesions caused by A. brassicicola. The antibodies did not prevent A. brassicicola infection on dewaxed cauliflower leaves, again indicating a crucial role for lipase in the penetration phase of the infection (Berto et al., 1999). The B. cinerea triacylglycerol lipase was partially sequenced (Comménil et al., 1999). The corresponding gene (Lip1) was cloned and targeted mutants were made by insertion of a hygromycin resistance cassette. Lip1-deficient B. cinerea mutants did not produce extracellular lipase under inducing conditions, but remained able to infect intact primary 10 General introduction leaves of Phaseolus vulgaris (H. Reis and M. Hahn,