Tercera Parte del Plan Comercial: Acción de Intervensión & Desarrollo de

Effectors can be defined as small secreted proteins that alter host cell

structure and function, facilitate infection and suppress or activate effector-triggered

immunity (van de Wouw and Howlett, 2011). Kamoun (2009) defined effectors as

“molecules that alter host cell structure and function, thereby facilitating infection (i.

e. virulence factors or toxins) and/or triggering defence responses (i.e. avirulence

factors or elicitors)”. Effectors can also be defined as “secreted proteins and other

molecules which allow plant-associated organisms to modulate plant defence

circuitry and enable colonization of plant tissue” (Hogenhoutet al, 2009). Brown and

his colleagues (2012) expanded that ‘effectors’ that modulate the interaction between

pathogenic microbes and hosts have been identified from all (mutualistic, biotrophic,

hemibiotrophic, necrotrophic and saprophytic) lifestyles”.

In this chapter the definition of effectors is used as by Hogenhout et al.

(2009). Characteristics listed in the first chapter (see Section 1.9) could be useful to

discover / identify novel effector genes applicable for the development of specific

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4.1.2 Characteristics of knownF. oxysporumeffectors

This section is predominantly focusses on effectors occur in F. oxysporum.

However, characteristics of other fungal and oomycete effectors, listed in Chapter 1,

need to be considered for the identification of novel candidate effectors.

Effector proteins can be classified based on whether they interact with their

target in the host apoplast or cytosol (Kamoun, 2008).SIXgenes ofF. oxysporumare

secreted during infection into the host apoplast, and are generally small (<300 amino

acids), cysteine rich (two or more) proteins with a type II secretion signal at the N-

terminal (Rep, 2005; van der Does and Rep, 2007). SIX genes have no database

representatives outside of the genusFusarium(Houtermanet al., 2007), and the only

sequence similarity between them was found in their promoter region, where a

transposable element, mimp1, is harboured in all SIX genes (Sarah Schmidt,

University of Amsterdam, pers. comm.).

Almost all known SIX genes of F. oxysporum f. sp. lycopersici (FOL) are

coded on a mobile, lineage specific chromosome (chromosome 14) which is rich in

transposable elements (Maet al., 2010). Similarly, the first conditionally dispensable

chromosomes encoding genes that are required for pathogenicity on pea, such as pea

pathogenicity (PEP1, PEP2, and PEP5 genes) and the pisatin demethylase (PDA1)

gene, were discovered in the genome ofFusarium solanif. sp.pisi(Hanet al. 2001).

The FOL effector geneSIX1(termed Fol-SIX1) is highly up-regulated during

colonisation and requires living plant cells as only 5–20% of hyphae express Fol-

SIX1when growing in tomato cell cultures (van der Doeset al., 2008). Expression of

the Fo5176-SIX4 homologue of the Arabidopsis pathogen F. oxysporum was 18- to

70- fold less when grown on PDA plates compare to that measured in planta

starvation (Snoeijers et al., 1999, Stephenson et al., 2000; Talbot et al., 1993)

although in F. oxysporum, no up-regulation of effector genes was detected during

nitrogen starvation of FOL (Divon et al., 2005), but a very low secretion was

observed when the fungus was grown on minimal medium with sucrose and KNO3

(van der Does and Rep, 2007).

Recently, two possible effector encoding genes, putative effector protein 1

(Fov-PEP1) and Fov-PEP2, have been discovered from F. oxysporum f. sp.

vasinfectum (FOV) (Chakrabarti et al., 2011). These genes were identified in a

cDNA library of 2100 clones isolated from cotton seedlings infected with FOV under

tissue-culture conditions (McFaddenet al., 2006) and selected on the basis of criteria

such as short, cysteine rich, secreted, had no database representatives at all or outside

of Fusarium genus and was only present in FOV isolates. A homologue of Fov-

PEP1is present in the FOL genome sequence as a single copy gene on chromosome

14, interrupted by a retrotransposon, whileFov-PEP2had no homologues in the FOL

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4.2 AIMS AND OBJECTIVES

The aims of this part of the project were:

 To obtain the whole genome sequence of a F. oxysporum f. sp. cepae(FOC) to identify candidate effector genes in a representative isolate

 To screen a set of candidate effector genes in a collection of F. oxysporum

isolates representing several formae speciales for the presence / absence.

 To compare the sequences of putative effector genes from different formae speciales

 Evaluate and test bioinformatics tools and markers for the identification of further effector genes

4.3 MATERIALS AND METHODS

4.3.1 Preparation of DNA samples for genome sequencing of

F.

oxysporumf. sp. cepae(FOC)

4.3.1.1 Description of FOC isolate chosen for genome sequencing

A very aggressive FOC strain, Fus2, was used for whole genome sequencing.

Fus2 was isolated from onion grown in Lincolnshire, UK and has been used to screen

onion cultivars for resistance and also to test effectiveness of biocontrol treatments

(Tayloret al., 2012; Ralph Noble, East Malling Research, pers. comm.).

4.3.1.2 DNA extraction

The CTAB extraction method (Li et al, 1994 modified by Dez Barbara,

University of Warwick) was employed to obtain good quality (unsheared) DNA for

genome sequencing. 250 mg of freeze dried mycelium of Fus2 was homogenised

with 1 g of sterile sand using a chilled, sterile pestle and mortar. The ground

mycelium/sand was transferred into a 50 ml Falcon tube and mixed by inversion with

15 ml extraction buffer (8.18g NaCl dissolved in 70 ml dH2O with 5 ml 2M pH 8.0

Tris-Cl, 4 ml 0.5M pH 8.0 EDTA, 7.4 ml 10% cetyltrimethylammonium bromide

(CTAB), 13.6 ml dH2O, 2 g polyvinylpyrrolidone 40 (PVP-40) and 0.5 ml 2-

mercaptoethanol). The sample was incubated at 60°C for 30 minutes and mixed

gently by inverting occasionally. 15 ml of the chloroform/penta-1-ol wash (96 ml

chloroform and 4 ml penta-1-ol) was added to the sample which was mixed by gentle

inversion for 10 minutes followed by centrifugation. All centrifugation steps were

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aqueous phase was transferred into a sterile 50 ml Falcon tube and 15 ml of

chloroform/penta-1-ol wash was added again followed by gentle mixing by inversion

for 10 minutes then centrifuged. The aqueous phase was transferred into a sterile 50

ml Falcon tube and centrifuged again. 0.6 volumes of cold (-20°C) isopropanol was

added to aqueous phase and mixed by inverting tube gently. The sample was

incubated at room temperature for 2 hours before centrifuging at 460g (1500 rpm) for

2 minutes at 20°C. The supernatant was then removed and the pellet dissolved in 10

ml wash buffer (66 ml 100% ethanol and 34 ml 0.1M NaCl) by gentle inversion for

20 minutes followed by centrifugation. Supernatant was removed and rinsed again in

10 ml wash buffer then was centrifuged. The supernatant was removed and the pellet

was air dried for 20 minutes. The pellet was then dissolved in 1.5 ml Tris-EDTA

(10mM pH 8.0 Tris-Cl and 1 mM EDTA) and transferred into a 1.5 ml Eppendorf

tube using a pipette tip with the end cut off. The sample was centrifuged at maximum

speed for 5 minutes and the supernatant was transferred into a 1.5 ml Eppendorf tube

using a pipette tip with the end cut off. The sample was quantified using a Nanodrop

1000 spectrophotometer (Thermo Scientific).

4.3.1.3 RNase treatment and purification of the nucleic acid extract

RNase treatment was applied to degrade any RNA in the sample.

Approximately 10µg of DNA was taken and 1 µl of RNase (Qiagen RNase A 7 U/µl)

added in a total volume of 25 µl. The sample was incubated at room temperature for

30 minutes then desalted using a Qiaex II gel extraction/desalting kit (Qiagen)

following the manufacturer’s guidelines with a minor modification. The sample was

eluted once in 30 µl of buffer EB from a QIAQuickPCR purification kit (Qiagen).

Scientific) then by Qubit fluorospectrometer using a broad range dsDNA kit

(Invitrogen). The DNA was diluted using buffer EB (Qiagen) to give a final volume

of 75 µl and a concentration of 20 ng/µl.

4.3.1.4 Verification of purity and quality of the DNA sample

Amplification of the ITS region (using ITS1 and ITS4 primer pair and

conditions published by Whiteet al., 1990) and theTEFgene (Chapter 2) were used

to ensure that the DNA sample is not contaminated and applicable for amplification.