Full Journal - Journal of Cell and Molecular Biology - Haliç Üniversitesi

More documents

Recommendations

Info

96 Figen Mert-Türk and Eric B. Holub Table 1: The sequences of primers encompassing RPP1 and RPP13 resistance genes their restriction enzymes (Nam et al., 1989; Oppenheimer et al., 1991; Bittner-Eddy et al., 1999 and 2000; Bittner-Eddy, personel communication). Primers R.E. a Gl-1 F b R c 5’-ATA TTG AGT ACT GCC TTT AG-3’ Taq1 5’-CCA TGA TCC GAA GAG ACT AT-3’ Py3003 F 5’-TCC TGT GTG TAG AGA ACC GC-3’ Rsa1 R 5’-TAG CGA GAA AAT AAT GTC TG-3’ pAT389 F 5’-AAT CAC CAT TAC TAA TCA GG-3’ n.e. d R 5’-AGA TTG GCA TCG TGA GGC AC-3’ m249 F 5’-CAG AGA GTG ACC AAA TCT GAA CC-3’ Aci1 R 5’-GCA TTA TGT TAG ACC AAT GTG C-3’ p1C7L F 5’-CTC CCC AAG TAG GCT TCC ATT C-3’ Mse1 R 5’-GAG TGT TGT TGG CTT TCA TGC AG-3’ a R.E. Restriction enzymes b F: Forward primers c R: Reverse primers d n.e. No Enzyme necessary as natural polymorphism is present Restriction enzyme digestion To identify restriction endonucleases that would reveal polymorphism in the amplified fragments, restriction digests were carried out in a final 20µl volume containing 12.5 µl of PCR product, 2 µl 10X enzyme buffer, 1-3 units of appropriate enzyme (0.2- 0.4µl) and 5.1-5.3 µl sterile ddH2O as shown in Table 1. The tubes were incubated at temperature advised by the manufacture. Electrophoresis Restriction fragments were assesed by electrophoresis on a 1-2.5% agarose gel made up in 1X TBA buffer; the same buffer was used as a running buffer. 1/5 volume of loading dye was added to each tube before loading into the wells of the gel. Electrophoresis was carried out at 85-150 V for 2-3 h. The gel then removed from the tank and submerged in a solution containing 0.5 µg/ml ethidium bromide in deionized water. The gel was destained in deionized water for 15 min and visulalized in UV transilluminator for polymorphism. Results Recombinants carrying Nd-alleles of RPP1, RPP13 and the pad3 allelle from the Col-pad3 mutant After testing 177 F2 plants using TLC plate assays, 37 F2 genotypes were identified as camalexin deficient when the plates were visualised under UV radiation suggesting that they were homozygous for pad3 (pad3 is a single recessive mutation, Glazebrook and Ausubel, 1994). Among these genotypes three (671, 678 and 848) were found homozygous for Nd-1 DNA across the interval containing RPP1 and RPP13. These genotypes were selfed until F4 seeds were obtained to minimise the heterozygosity in other loci. F4 seeds were, therefore, used for the pathogenicity test using the isolates of P. parasitica (Emoy2, Waco5 and Hiks1 for RPP1 resistance gene; Edco1, Maks9, Aswa1, Goco1 and Emco5 for RPP13 resistance gene). All three Nd-pad3 genotypes (671, 678 and 848) were found to be homozygous in RPP1 and RPP13 genes from Nd-1 and pad3 allele from the Col-pad3 mutant. All these genotypes were included in the
Table 2: Phenotypic interaction between the F4 genotypes of Arabidospsis thaliana carrying Nd-1 resistance genes at RPP1, RPP13 and the pad3 allele from Col-pad3 mutant following inoculation with isolates of Peronospora parasitica. Lines of Predicted A. thaliana Genotype a Nd-1 RPP1/RPP13/PAD3 PN b experiment with a thought that all three may have different genotypes as a result of crossing-over following cross-pollination. No camalexin accumulation was observed in these genotypes during screening, as all genotypes were selected for camalexin deficiency. Interaction phenotypes (IPs) of the genotypes following inoculation by the isolates recognised by RPP1 and RPP13 genes Three genotypes (hereafter will be abbreviated as Ndpad3) containing pad3 mutation along with RPP1 and RPP13 resistance genes, the Col-pad3 mutant and its wild-type Col-0, where it was derived from, and Nd- 1 seedlings were inoculated by the isolates of P. parasitica at 7 days after sowings. The seedlings were assessed for their phenotypes 3 and 7 days after inoculation (dai). The Nd-1 seedlings exhibited pitting necrosis and no sporulation of all three isolates were observed (Figure 1a). Following inoculation by Waco5, Emoy2 and Hiks1 (RPP1-avirulence isolates), the wild-type Col-0 exhibited heavy sporulation, low sporulation with flecking host response and no sporulation with flecking response, respectively. The Col-pad3 seedling were also as susceptible as wild-type Col-0 after inoculation with Waco5, however, exhibited medium sporulation with the isolate Emoy2 (Figure 1b). Although Hiks1 was Camalexin and RPP1 and RPP13 resistance genes 97 Isolates of Peronospora parasitica RPP1-avirulence RPP13- virulence Waco5 Emoy2 Hiks1 Edco1 Maks9 Aswa1 Goco1 Emco5 PN PN FN FN FN FN FN Col-0 rpp1/rpp13/PAD3 H FL FN H H H H H Col-pad3 rpp1/rpp13/pad3 H M N H H H H H Nd-pad3 (671) RPP1/RPP13/pad3 PN PN PN FN FN FN FN FN Nd-pad3 (678) RPP1/RPP13/pad3 PN PN PN FN FN FN FN FN Nd-pad3 (848) RPP1/RPP13/pad3 PN PN PN FN FN FN FN FN a Based on genetic marker assisted selection in the case of Nd-pad3 mutants numbered 671, 678 and 848. b Interaction phenotypes (per cotyledon). H: heavy sporulation (>20 conidiophores); M: medium sporulation (5-20 conidiophores); L: low sporulation (1-5 conidiophores); N: none sporulation; F: chlorotic flecks; P: necrotic pits. not able to grow in the Col-pad3 mutant, the host response of the mutant was different than wild type as no flecking response, which is associated with HR, was observed. All three Nd-pad3 genotypes exhibited a PN phenotype following inoculation with RPP1avirulent isolates (Emoy2, Waco5 and Hiks1) as shown in Figure 1c and Table 2. A. thaliana accession Nd-1 carries RPP13 gene that recognises avirulence product of at least five isolates of P. parasitica (Edco1, Maks9, Aswa1, Goco1 and Emco5). This gene has recently been cloned (Bittner-Eddy et al., 2000). Clorotic flecking host response with no sporulation of the pathogen was seen in the accession Nd-1 (Figure 1d). However, the Col-0 accession does not have this allele, therefore, the plants are very susceptible to the isolates which are recognised by RPP13 gene. The Col-pad3 mutant was also suseptible to these isolates (Figure 1e). All three genotypes were, however, showed the same response as seen in Nd-1 parent. The seedlings were resistant to the five isolates and flecking response was seen 7 dai. No sporulation of the pathogen was seen (Figure 1f and Table 2). Discussion The existence of pad3 mutation on Nd-1 background did not obviously affect the function of either RPP genes.
Page 1 and 2: STANBUL 1998 VOLUME 1 • NO. 2 •
Page 3 and 4: Journal of Cell and Molecular Biolo
Page 5 and 6: 46 Gülriz Bayçu Cd Contamination
Page 7 and 8: 48 Gülriz Bayçu Figure 2: Optimiz
Page 9 and 10: 50 Gülriz Bayçu PC production was
Page 11 and 12: 52 Gülriz Bayçu plants and fungi
Page 13 and 14: 54 Gülriz Bayçu References Abraha
Page 17 and 18: cells (cancer), inflammatory sites
Page 19 and 20: 1991, Russel and Synder, 1968; Pegg
Page 21 and 22: Figure 3: Several sources of PAs ar
Page 23 and 24: chemotherapy and lead to a very lar
Page 25 and 26: Thomas T and Thomas T. Polyamines i
Page 27 and 28: 70 Tülin Aktaç and Elvan Bakar Ma
Page 29 and 30: 72 Tülin Aktaç and Elvan Bakar Ak
Page 31 and 32: 74 Seyhan Altun et al. Altun (1996)
Page 33 and 34: 76 Seyhan Altun et al. increased a
Page 35 and 36: 78 Seyhan Altun et al. PH ratio and
Page 39 and 40: epirubicin + tamoxifen were added t
Page 41 and 42: Murphy B and Muss HB. Hormonal ther
Page 43 and 44: 88 Gülruh Ulako¤lu et al., 1993).
Page 45 and 46: 90 Gülruh Ulako¤lu According to t
Page 49: pad3 locus from the Col-pad3 mutant
Page 53 and 54: locus recognise distinct Peronospor
Page 55 and 56: 102 Narçin Palavan-Ünsal et al. I
Page 57 and 58: 104 Narçin Palavan-Ünsal et al. F
Page 59 and 60: 106 Narçin Palavan-Ünsal et al. F
Page 61 and 62: 108 Narçin Palavan-Ünsal et al. c
Page 63 and 64: 110 Ayfle TABAKO⁄LU-O⁄UZ, Anima
Page 65 and 66: 112 by superscript small letters an
Page 67 and 68: 114 Volume 1, No. 2 Review articles
Page 69: Journal of Cell and Molecular Biolo

Full Journal - Journal of Cell and Molecular Biology - Haliç Üniversitesi

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?