The Botrytis cinerea endopolygalacturonase gene family

C ell w all d egrading e nzyme s (CWDEs) secreted by microbial plant pathogens have been suggested to function as virulence factors. Evidence that particular bacterial CWDEs contribute to virulence has emerged in the last two decades. Targeted gene replacement of different genes encoding CWDEs resulted in mutants with reduced virulence on a number of host plants. Similar molecular genetic approaches in plant pathogenic fungi have, until recently, been unsuccessful in elucidating a role for fungal CWDEs in pathogenesis. This thesis describes molecular genetic analyses of CWDEs secreted by the necrotrophic plant pathogenic fungus Botrytis cinerea , the causal agent of gray mould.From literature it was known that B. cinerea secretes many CWDEs when grown in liquid culture. The number of CWDE encoding genes present in the B. cinerea genome was unknown and detailed expression studies were lacking. In order to fill this knowledge gap we used the following strategy:Cloning of genes encoding CWDEsStudy of the expression of CWDE genes both in liquid cultures and in plantaTargeted deletion of CWDE genes that have expression patterns that indicate a function in the infection processChapter 1 introduces the research area and gives an outline of the thesis. It describes a model of the chemical and structural composition of the plant cell wall and reviews various classes of microbial CWDEs. It summarises previously published data on the role of bacterial and fungal CWDEs in pathogenesis in general and on the CWDEs secreted by B. cinerea in particular. B. cinerea has a wide host range but prefers hosts that contain high amounts of pectin. Therefore the focus was on endo p oly g alacturonases (endoPGs), enzymes that cleave homogalacturonan, a major constituent of pectin.In order to study gene expression of B. cinereain planta , it was essential to develop a standardised inoculation procedure that enables reproducible infections both in time and space. The development of this inoculation procedure for tomato leaves is described in Chapter 2. The expression of two fungal genes and a number of plant PR-protein genes was investigated in time course experiments performed at two different incubation temperatures.Subsequently, we set out to clone the genes of interest, analysed their expression and studied the effect in pathogenesis by targeted gene replacement. The genes were isolated by hybridisation with heterologous probes. The first gene that was cloned and characterised, Bcpg 1, is constitutively expressed. Targeted replacement of this gene resulted in a mutant with reduced virulence on apple fruits and tomato (Chapter 3). Subsequently, five additional endoPG genes were isolated (Chapter 4). The gene products were compared with other fungal endoPGs and it was shown that the members of the B. cinereaBcpg gene family fall into at least three distinct monophyletic groups (Chapter 4).The members of the endoPG gene family, denoted as Bcpg 1-6, are differentially expressed in liquid cultures that differed in carbon source or pH (Chapters 4). The constitutive expression pattern of Bcpg 1, as found in Chapter 3, was further confirmed. Bcpg 2 is expressed under all circumstances tested except when B. cinerea is grown in glucose-containing medium at low pH. Bcpg 3 is expressed at low ambient pH. Bcpg 4 is induced by the pectin breakdown end-product galacturonic acid, and is repressed by glucose. Bcpg 5 expression can be induced by a yet unknown factor present in apple pectin. Bcpg 6 is, like Bcpg 4, induced by galacturonic acid but is, unlike Bcpg 4, not repressed by glucose. The expression of the endoPG gene family enables the fungus to degrade pectate in a flexible manner. It enables the fungus to respond to environmental signals like nutrient availability and pH.The expression of the endoPG gene family during infection of tomato leaf, broad bean leaf, apple fruit and courgette fruit was studied (Chapter 5). Expression of the genes in planta is differential and most expression patterns can be explained by the results of expression studies in liquid cultures. Bcpg 1 is expressed in all host tissues tested, whereas expression of Bcpg 2 is evident in tomato, broad bean and courgette. Bcpg 3 and Bcpg 5 are expressed in apple fruit. Bcpg 4 and Bcpg 6 are expressed in all host tissues tested.Chapter 6 discusses the results in a broader context. It is hypothesised that, besides Bcpg 1, additional members of the Bcpg gene family contribute to virulence, albeit likely under specific circumstances. It is suggested that fungal CWDEs can play a role in plant pathogenesis but that this role also strongly depends on the lifestyle of the fungus. It is postulated that B. cinerea depends strongly on endoPGs for successful infection. The research described in this thesis may lead to novel disease control strategies that rely on P oly G alacturonase I nhibiting P rotein (PGIP) expression in transgenic host plants.