The Botrytis cinerea endopolygalacturonase gene family.

TEN HAVE A

Año: 2000 p. 119

90-5808-227-X

Cell wall degrading enzymes (CWDEs) secreted by microbial plant pathogens have beensuggested to function as virulence factors. Evidence that particular bacterial CWDEscontribute to virulence has emerged in the last two decades. Targeted gene replacement ofdifferent genes encoding CWDEs resulted in mutants with reduced virulence on a number ofhost plants. Similar molecular genetic approaches in plant pathogenic fungi have, untilrecently, been unsuccessful in elucidating a role for fungal CWDEs in pathogenesis. Thisthesis describes molecular genetic analyses of CWDEs secreted by the necrotrophic plantpathogenic fungus Botrytis cinerea, the causal agent of gray mould.         From literature it was known that B. cinerea secretes many CWDEs when grown inliquid culture. The number of CWDE encoding genes present in the B. cinerea genome wasunknown and detailed expression studies were lacking. In order to fill this knowledge gap weused the following strategy:    (1) Cloning of genes encoding CWDEs    (2) Study of the expression of CWDE genes both in liquid cultures and in planta    (3) Targeted deletion of CWDE genes that have expression patterns that indicate a    function in the infection process         Chapter 1 introduces the research area and gives an outline of the thesis. It describesa model of the chemical and structural composition of the plant cell wall and reviews variousclasses of microbial CWDEs. It summarises previously published data on the role of bacterialand fungal CWDEs in pathogenesis in general and on the CWDEs secreted by B. cinerea inparticular. B. cinerea has a wide host range but prefers hosts that contain high amounts ofpectin. Therefore the focus was on endopolygalacturonases (endoPGs), enzymes that cleavehomogalacturonan, a major constituent of pectin.         In order to study gene expression of B. cinerea in planta, it was essential to develop astandardised inoculation procedure that enables reproducible infections both in time andspace. The development of this inoculation procedure for tomato leaves is described inChapter 2. The expression of two fungal genes and a number of plant PR-protein genes wasinvestigated in time course experiments performed at two different incubation temperatures.         Subsequently, we set out to clone the genes of interest, analysed their expressionand studied the effect in pathogenesis by targeted gene replacement. The genes wereisolated by hybridisation with heterologous probes. The first gene that was cloned andcharacterised, Bcpg1, is constitutively expressed. Targeted replacement of this gene resultedin a mutant with reduced virulence on apple fruits and tomato (Chapter 3). Subsequently, fiveadditional endoPG genes were isolated (Chapter 4). The gene products were compared withother fungal endoPGs and it was shown that the members of the B. cinerea Bcpg gene familyfall into at least three distinct monophyletic groups (Chapter 4).          The members of the endoPG gene family, denoted as Bcpg1-6, are differentiallyexpressed in liquid cultures that differed in carbon source or pH (Chapters 4). The constitutiveexpression pattern of Bcpg1, as found in Chapter 3, was further confirmed. Bcpg2 isexpressed under all circumstances tested except when B. cinerea is grown in glucose-containing medium at low pH. Bcpg3 is expressed at low ambient pH. Bcpg4 is induced bythe pectin breakdown end-product galacturonic acid, and is repressed by glucose. Bcpg5expression can be induced by a yet unknown factor present in apple pectin. Bcpg6 is, likeBcpg4, induced by galacturonic acid but is, unlike Bcpg4, not repressed by glucose. Theexpression of the endoPG gene family enables the fungus to degrade pectate in a flexiblemanner. It enables the fungus to respond to environmental signals like nutrient availability andpH.          The expression of the endoPG gene family during infection of tomato leaf, broadbean leaf, apple fruit and courgette fruit was studied (Chapter 5). Expression of the genes inplanta is differential and most expression patterns can be explained by the results ofexpression studies in liquid cultures. Bcpg1 is expressed in all host tissues tested, whereasexpression of Bcpg2 is evident in tomato, broad bean and courgette. Bcpg3 and Bcpg5 areexpressed in apple fruit. Bcpg4 and Bcpg6 are expressed in all host tissues tested.          Chapter 6 discusses the results in a broader context. It is hypothesised that, besidesBcpg1, additional members of the Bcpg gene family contribute to virulence, albeit likely underspecific circumstances. It is suggested that fungal CWDEs can play a role in plantpathogenesis but that this role also strongly depends on the lifestyle of the fungus. It ispostulated that B. cinerea depends strongly on endoPGs for successful infection. Theresearch described in this thesis may lead to novel disease control strategies that rely onPolyGalacturonase Inhibiting Protein (PGIP) expression in transgenic host plants.