Insertional mutagenesis in the vascular wilt pathogen Verticillium dahliae

Vascular wilt diseases caused by soil-borne pathogens are among the most devastating plant diseases worldwide. The ascomycete fungus Verticillium dahliae causes vascular wilt diseases in hundreds of dicotyledonous plant species, including important crops such as eggplant, lettuce, olive, spinach and tomato. The resting structures, microsclerotia, are triggered by root exudates to germinate and penetrate the roots after which the fungus grows into the xylem vessels. The fungus colonizes these vessels and interferes with the transportation of water and nutrients, resulting in the development of symptoms such as stunting, wilting, chlorosis and vascular browning. Verticillium wilt diseases are difficult to control due to the longevity of the microsclerotia, the broad host range of the pathogen, the inability of fungicides to kill the fungus once it has colonized the xylem vessels and the lack of natural resistance in many plant species. Chapter 1 is the introduction to this thesis that describes the identified pathogenicity and virulence factors of V. dahliae and strategies to identify these components. In spite of the economic importance of V. dahliae, relatively few pathogenicity genes have been identified in this species. With the availability of whole genome sequences and the development of functional genomics tools such as random mutagenesis, targeted mutagenesis, transcriptomics, RNA interference (RNAi) and comparative genomics, more strategies have become available to identify novel pathogenicity and virulence genes. Chapter 2 focuses on the identification of virulence and pathogenicity genes of V. dahliae by screening of a library of random T-DNA insertion mutants. Using Agrobacterium tumefaciens-mediated transformation, 900 T-DNA transformants with random insertions were generated and screened for altered virulence on susceptible tomato plants. This screening, followed by inverse PCR on selected transformants, resulted in the identification of 55 potential pathogenicity and virulence genes. One of the potential pathogenicity genes, VdNRS/ER, is a homolog of a nucleotide-rhamnose synthase/epimerase-reductase (NRS/ER), which is presumably involved in the biosynthesis of UDP-rhamnose. Using targeted mutagenesis, VdNRS/ER was deleted from wild-type V. dahliae and the resulting deletion mutants were characterized. VdNRS/ER deletion mutants exhibit unaltered vegetative growth and sporulation, but the deletion mutants were no longer pathogenic on tomato and N. benthamiana and showed impaired root attachment on tomato seedlings. These data suggest that UDPrhamnose is required for pathogenesis of V. dahliae. Chapter 3 describes the role of the V. dahliae homolog of Sge1, a transcriptional regulator that was shown to play a role in pathogenicity and regulate effector gene expression in Fusarium oxysporum. In this chapter it is demonstrated that V. dahliae Sge1 (VdSge1) is required for radial growth and production of asexual conidiospores. It is furthermore shown that VdSge1 deletion strains have lost pathogenicity on tomato. Since the VdSge1 deletion mutants are not able to infect and colonize tomato plants, a tomato cell suspension culture was used to the study the expression of Ave1, as well as nine other genes of which the expression is highly induced in planta. This assay revealed that VdSge1 is not required for the induction of the Ave1 effector that activates resistance mediated by Ve1 in tomato. Furthermore, the expression of one other putative effector gene was not affected by VdSge1 deletion. However, VdSge1 was shown to be required for the expression of six putative effector genes, whereas expression of the remaining two putative effectors genes was negatively regulated. Thus, the data show that VdSge1 is required for V. dahliae pathogenicity and differentially regulates effector gene expression. Chapter 4 describes the functional characterization of the gene family encoding necrosis- and ethylene-inducing-like proteins (NLPs) of V. dahliae. The cytotoxic activity of NLP family members was determined using agroinfiltration into tobacco leaves. This resulted in the identification of two out of the seven NLPs, VdNLP1 and VdNLP2, that induced plant cell death. The genes encoding these cytotoxic NLPs were found to be induced in V. dahliae upon colonization of tomato. Targeted deletion of VdNLP1 and VdNLP2 significantly reduced the virulence of V. dahliae on tomato and Arabidopsis plants. In contrast, only deletion of VdNLP1 affected virulence on N. benthamiana whereas deletion of NLP2 did not. However, subsequent transcriptional analysis revealed that VdNLP2 was not expressed in V. dahliae during colonization of N. benthamiana. Moreover, VdNLP2 also affects vegetative growth and conidiospore production. In conclusion, the expanded V. dahliae NLP family shows differential cytotoxic activity between family members and in planta induction of the cytotoxic NLP genes varies between plant hosts. In addition, VdNLP2 plays a role in vegetative growth and conidiospore production in addition to its contribution to virulence. Thus, evidence is provided for functional diversification within the V. dahliae NLP family. Finally in Chapter 5, the major findings of this thesis are discussed and placed in a broader perspective.