Identificar el estado actual de incorporación de criterios de responsabilidad

The odd-ler genomic region required for the autonecrosis was mapped to a ~52 kb region on

Chromosome 3 in the reference genome sequence of Col-0 (Shirzadian-Khorramabad et al., 2010) (Appendix 5). In Col-0, this region contains a cyclophillin protein coding gene At3g44600, a serine/threonine protein kinase coding gene AT3G44610, a protein tyrosine

phosphatase coding gene-At3g44620 , 2 RESISTANCE TO PERENOSPORA PARASITICA 1

(RPP1) like disease resistance R genes-At3g44630 and At3g44670, a histone deacetylase coding gene-At3g44660 and three transposable elements (At3g44- 605, -640 and -650) (Appendix 5).

To identify potential candidates for the odd-ler gene, the protein coding genes in the previously mapped odd-ler region were sequenced. Sequencing of the At3g44610 and At3g44620 in the odd-ler region showed significant nucleotide variation leading towards various amino acid substitutions in the putative protein-coding gene models (Appendix 6). The primers that were designed to amplify the disease resistance R gene At3g44630, on the basis of genome sequence map in Col-0, did not amplify any gene fragment from Ler-0 but were successful in amplifying the target gene from Col-0. This indicated polymorphism(s) in the Ler-0 At3g44630 gene and therefore multiple primer sets were designed to amplify the gene in overlapping fragments. Using these primer sets, various genetic fragments were amplified. Unfortunately, these fragments showed extensive polymorphism in the R gene sequence and failed to overlap with each other (Appendix 6). This suggested multiple or highly diverged RPP1-like R gene(s) at the odd-ler region in comparison to the odd-col region. As R genes evolve rapidly in comparison to other genes (Meyers et al., 2005), the genetic variation observed in the odd-ler region was likely caused by the duplication, deletion or insertion of the R genes. Regardless, the At3G44610 (serine-threonine protein kinase), At3G44620 (encoding a protein tyrosine phosphatase) and the highly variant RPP1-like R gene(s) were identified as candidate odd-ler genes.

An attempt was made to silence these genes in the old3-1 mutant using an RNAi approach. Transformation of the RNAi construct aimed for targeting the At3g44610 or At3g44620 gene in

the old3-1 mutant did not rescue the phenotype at 21 ºC (Figure 4.5). For RNAi-mediated

and B. These constructs targeted RPP1-specific sequences in the N and C terminal part of the

RPP1 protein-coding genes on chromosome 3 (See methods). Targeting of the RPP1-like R

genes only, employing two independent constructs, rescued the lethal phenotype of the old3-1 mutants (Figure 4.5). The rescue of the phenotype was consistent with the decrease in expression of the RPP1-like R gene(s), tested using a conserved RPP1 specific marker (Figure 4.5).

Further the mapped odd-ler region was also found to overlap with Quantitative Trait Locus 3 (QTL3) which contains a cluster of 8 RPP1-like disease resistance (R) genes (Alcázar et al., 2009) in contrast to the two R genes present in Col-0 (Appendix 6). This confirmed the nature of

R gene polymorphisms in the odd-ler region. Transcript accumulation of the RPP1-like R genes

in the odd-ler region was therefore determined in the old3-1 mutants and rescued lines in a temperature shift experiment. Gene-specific primers were designed to the putative coding regions and tested for their ability to detect the expression of each R gene specifically. Measurements of the relative gene expression of RPP1-like R alleles using qRT-PCR showed that the transcript abundance of RPP1-like R1, R2, R3, R4, R5, R7 and R8 genes was up- regulated in the old3-1 mutant, 24 hr post-temperature shift from 28 to 21ºC (Figure 4.6). The transcript accumulation of the truncated R6t allele was however not determined. In comparison, the rescued old3-1 mutant line B40-12 transformed with RNAi-RPP1 construct, showed decreased transcript accumulation of all R genes at either growth temperature (Figure 4.6).

Figure 4.5. RPP1 gene(s) show negative epistasis to the old3-1 mutation. (A) shows phenotypes of the old3-1 mutant, transgenic old3-1 mutant and wild-type Ler-0. Plants were grown for 21 days at 21 °C. Representative plants are shown. Bar indicates 5 mm. (B) shows the expression of RPP1-like R genes in the old3-1 mutant and three T3 homozygous independent transgenic lines from construct B. Plants were grown for 24 days at 21 ºC and harvested for analysis. Data shows mRNA abundance of RPP1-like R genes, using an RPP1 cDNA specific marker, relative to Actin2. Data are the mean of two biological replicates and each biological replicate is the mean of 3 technical replicates. Error bars represent standard deviation of the mean.

Figure 4.6. Transcript abundance of RPP1-like R genes present in the odd-ler region is affected upon activation of autonecrosis in the old3-1 mutant. Figure shows the quantification of the expression of full-length RPP1-like R gene(s) in the old3-1 mutant and the rescued line B40-12. Relative expression was measured by qRT-PCR using gene-specific primers at 0 and 24 hr after temperature shift from 28 to 21°C. Data represents relative expression levels of each R gene to the expression values of Actin2. The data represents mean of 3 biological replicates at each time point. Error bars represents standard deviation. Statistically significant differences in the value of the gene expression between transgenic rescued B40-12 and the old3-1 mutant using Student’s t test are shown by * at P< 0.05, ** at P< 0.01 and *** at P< 0.005.