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.
|Qualification||Doctor of Philosophy|
|Award date||20 Jan 2014|
|Place of Publication||Wageningen|
|Publication status||Published - 2014|
- plant pathogenic fungi
- verticillium dahliae
- insertional mutagenesis
- solanum lycopersicum
- gene mapping