Biological material - Materials and Methods

2. Materials and Methods

2.1. Biological material

Bacterial and fungal strains and plant material used in this study are listed in Table 2.1. Plasmids and cosmids are listed in Table 2.2.

Table 2.1: Bacterial strains, fungal strains and plant material

Strain Relevant Characteristics Source or Reference

Fungal Strains E. festucae

PN2278 Wild-type Fl1 Young et al. (2005)

PN2656 ∆sakA; Fl1/∆sakA::PtrpC-hph; HygR_(T1-3) _{Eaton et al. (2008)} PN2657 PN2278/pCE12; HygR_(T25-2) _{Eaton et al. (2008)} PN2658 ∆sakA/sakA; PN2656/pCE1; pII99; HygR_{; Gen}R_{(C5-2) Eaton et al. (2008)} PN2660 ∆pakA; Fl1/∆pakA::PtrpC-hph; HygR _{This study} PN2661 ∆pakB; Fl1/∆pakB::PtrpC-hph; HygR _{This study} PN2697 ∆pakA/pakA; PN2660/pCE43; HygR_{; Gen}R _{This study} PN2698 ∆pakB/pakB; PN2661/pCE42; HygR_{; Gen}R _{This study}

PN2327 ∆noxA; Fl1/∆noxA::PtrpC-hph; HygR _{Tanaka et al. (2006)} PN2479 ∆noxR; Fl1/∆noxR::PtrpC-hph; HygR _{Takemoto et al.}

(2006)

S. pombe

PN2578 (WT) WT; 972 h- Eaton et al. (2008)

PN2580 (JM1745) sty1Δ; sty1::ura4 leu1.32 ura4-D18, h- J. Millar, UK

PN2582 PN2580/pREP81 Eaton et al. (2008)

PN2584 PN2580/pF37 Eaton et al. (2008)

PN2585 PN2580/pCE22 Eaton et al. (2008)

PN2586 PN2580/pCE23 Eaton et al. (2008)

PN2606 PN2580/pREP81-Cgfp Eaton et al. (2008)

PN2607 PN2580/pCE30 Eaton et al. (2008)

E. coli Strains

XL-1 Blue supE44, hsdR17, recA1, endA1, gyrA46, thi, relA1, lac-_{, F’} [proAB+_,_lac_Iq_,_lac_Z_∆_{M15, Tn10 (Tet}R_)]

Bullock et al. (1987) Mach1 ∆recA1398, endA1, tonAΦ81, lacZ∆M15, ∆lacX74,

hsdR(rk- mk+)

TOP10 F–_, _mcrA_, _Δ₍_{mrr-hsdRMS-mcrBC}_), _φ₈₀_lacZΔ_M15, ΔlacX74, deoR, recA1, araD139, Δ(ara-leu)7697 galU, galK, rpsL(StrR_),_endA1_,_nupG

Invitrogen

DH5α F–_,_φ₈₀_lacZ_,_Δ_M15,_Δ₍_lacZYA_-_argF_{), U169,}_recA1_,_endA1_, hsdR17 (rk–, mk–), phoA, supE44, λ–, thi-1, gyrA96, relA1

Invitrogen

PN1908 Mach1/pCE1 Eaton et al. (2008)

PN1952 XL-1 Blue/pCE12 Eaton et al. (2008)

PN1972 XL-1 Blue/pCE22 Eaton et al. (2008)

PN1973 XL-1 Blue/pCE23 Eaton et al. (2008)

PN4020 XL-1 Blue/pCE30 Eaton et al. (2008)

PN1932 XL-1 Blue/pF37 Eaton et al. (2008)

PN1933 XL-1 Blue/pREP81 Eaton et al. (2008)

PN4019 XL-1/pREP81-Cgfp Eaton et al. (2008)

PN4047 DH5α/pCE35 This study

PN4048 DH5α/pCE36 This study

PN4050 DH5α/pCE37 This study

PN4051 DH5α/pCE38 This study

PN4052 DH5α/pCE40 This study

PN4053 DH5α/pCE41 This study

PN4081 TOP10/pCE42 This study

PN4082 TOP10/pCE43 This study

Plant Material

∆sakA infection analysis Round 1

G2869 L. perenne cv. Samson/PN2278; WT Fl1 This study G2870 L. perenne cv. Samson/PN2656 ; ΔsakA This study

G2871 L. perenne cv. Samson/PN2657 This study

G2872 L. perenne cv. Samson/PN2658 ; ΔsakA/sakA This study

Round 2

G3214, 3217, 3220, 3223, 3226

L. perenne cv. Samson/PN2278; WT Fl1 This study G3215, 3218, 3221,

3224, 3227

L. perenne cv. Samson/PN2656 ; ΔsakA This study G3216, 3219, 3222,

3225, 3228

L. perenne cv. Samson/PN2658 ; ΔsakA/sakA This study

Round 3

G3309 L. perenne cv. Samson/PN2278; WT Fl1 This study G3310-3312 L. perenne cv. Samson/PN2656 ; ΔsakA This study

G3313 L. perenne cv. Samson/PN2658 ; ΔsakA/sakA This study

Round 4

G3765-67; 3809-11 L. perenne cv. Samson/PN2278; WT Fl1 This study G3774-76; 3818-20 L. perenne cv. Samson/PN2656 ; ΔsakA This study G3777-79; 3821-23 L. perenne cv. Samson/PN2658 ; ΔsakA/sakA This study

∆pakA survival analysis

G3756-58; 3765-67; 3780-82; 3809-11; 3284-26

L. perenne cv. Samson/PN2278; WT Fl1 This study

G3759-61; 3768-70; 3783-85; 3812-14; 3827-29

L. perenne cv. Samson/PN2660; ΔpakA This study

G3762-64; 3771-73; 3786-88; 3815-17; 3830-32

L. perenne cv. Samson/PN2661; ΔpakB This study

Grown under sterile conditions

M59-63, 134-145 L. perenne cv. Samson/PN2278; WT Fl1 This study M39-58, 146-174 L. perenne cv. Samson/PN2656 ; ΔsakA This study M64-68 L. perenne cv. Samson/PN2658 ; ΔsakA/sakA This study

Table 2.2: Plasmids and cosmids

Name Relevant characteristics Source/reference Cosmid

44D6 pMO-cosX clone Fl1 genomic DNA cosmid library containing the sakA gene

Eaton et al. (2008) 24B3 pMO-cosX clone Fl1 genomic DNA cosmid library

containing the pakA gene

This study 9H6 pMO-cosX clone Fl1 genomic DNA cosmid library

containing the pakB gene

This study

Plasmid

pCE1 12 kb BamHI fragment ex cosmid clone 44D6 in pUC118, AmpR

Eaton et al. (2008) pCE12 3.2 kb EcoRI fragment ex pCE1 and 1.5 kb

sak22/sak23 PCR fragment in pSF15.15

Eaton et al. (2008) pCE22 1.7 kb sak24/sak39 PCR fragment in pREP81 Eaton et al. (2008) pCE23 1.1 kb sak24/sak39 cDNA fragment in pREP81 Eaton et al. (2008) pCE30 1.1 kb sak24/sak57 cDNA fragment in pREP81-Cgfp Eaton et al. (2008) pCE35 2 kb pak3/pak4 PCR fragment in pSF15.15 This study

pCE36 2.1 kb pak1/pak2 PCR fragment in pCE35 This study pCE37 1.6 kb pak7/pak8 PCR fragment in pSF15.15 This study pCE38 3 kb pak5/pak6 PCR fragment in pCE37 This study pCE42 8.3 kb BglII fragment ex 9H6 in pSF17.8 This study pCE43 4.9 kb pak22/pak23 PCR fragment in pCR4-Topo This study pF37 pREP81-sty1 HA6H J. Millar, UK pREP81 AmpR_;_LEU2 _{D. Mulvihill, UK} pREP81-Cgfp pREP81 with C-terminal GFP tag I. Hagan, UK pPN83 pBlueScriptII®_{KS(+) with P}_gpd_-EGFP-T_trpC_{; Amp}R _{Takemoto et al. (2006)} pII99 PtrpC-nptII-TtrpC ; AmpR_/GenR _{Namiki et al. (2001)}

pSF15.15 AmpR_{; Hyg}R _{S. Foster}

pSF17.8 AmpR_{; Gen}R _{S. Foster}

pUC118 AmpR Vieira & Messing (1987)

pGEM-T Easy AmpR _Promega

pCR4-Topo KanR _Invitrogen

In document Investigation of signalling involved in maintaining the mutually beneficial association between Epichloe festucae and perennial ryegrass : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at M (Page 51-55)