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The In Planta-Produced Extracellular Proteins ECP1 and ECP2 of Cladosporium fulvum Are Virulence Factors

¹Department of Phytopathology, Wageningen Agricultural University, Binnenhaven 9, 6709 PD, Wagenin-gen, The Netherlands; ²Institut des Sciences Végétales, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette Cedex, France; ³Department of Genetics, Wageningen Agricultural University, Drei-jenlaan 2, 6703 HA, Wageningen, The Netherlands

The two extracellular proteins ECP1 and ECP2 are abundantly secreted by the plant-pathogenic fungus Cladosporium fulvum during colonization of the intercellular space of tomato leaves. We examined the involvement of both proteins in pathogenicity and virulence of this fungus. ECP1-deficient, ECP2-deficient, and ECP1/ECP2- deficient isogenic C. fulvum strains were created by targeted gene replacement. Upon inoculation onto susceptible 6-week-old tomato plants, all three mutants showed reduced virulence. Deficiency in ECP2 resulted in a strain that poorly colonized the leaf tissue and secreted lower amounts of the in planta-produced ECP3, AVR4, and AVR9 proteins than the wild-type strain. The ECP2-deficient strain produced little emerging mycelium and few conidia. Deficiency in ECP1 did not significantly modify colonization of the leaf tissue, but reduced secretion of in planta-produced proteins. The ECP1-deficient strain emerged from stomata of the lower epidermis, but failed to sporulate as abundantly as the wild-type strain. A strain deficient in both ECP1 and ECP2 proteins had a phenotype similar to that of the ECP2-deficient strain. Accumulation of pathogenesis-related proteins and induction of late responses, such as leaf desiccation and abscission, occurred more quickly and more severely in tomato after inoculation with the ECP1-, ECP2-, and ECP1/ECP2-deficient strains than after inoculation with the wild-type strain. Moreover, partial collapse of stomatal guard cells occurred at emergence of the ECP2-deficient strain. These results indicate that the ECP1 and ECP2 proteins play a role in virulence of C. fulvum on tomato and suggest that both are involved in suppression of host defense responses.