Insertional mutagenesis in the vascular wilt pathogen Verticillium dahliae

P. Santhanam

Research output: Thesisinternal PhD, WU

Abstract

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.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Thomma, Bart, Promotor
  • de Wit, Pierre, Co-promotor
Award date20 Jan 2014
Place of PublicationWageningen
Publisher
Print ISBNs9789461738257
Publication statusPublished - 2014

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Keywords

  • plant pathogenic fungi
  • verticillium dahliae
  • wilts
  • insertional mutagenesis
  • solanum lycopersicum
  • tomatoes
  • models
  • pathogenicity
  • gene mapping
  • genomics

Cite this

Santhanam, P. (2014). Insertional mutagenesis in the vascular wilt pathogen Verticillium dahliae. Wageningen: Wageningen University.