Pectin methyl-esterases (PMEs) in wheat: genome-wide characterization and their role in wheat-Fusarium graminearum interaction
Author(s)
Zega, Alessandra
Date Issued
June 29, 2016
Type
Doctoral Thesis
Abstract
Pectin methyl esterase (PME) genes code for enzymes that are involved in structural modifications of the plant cell wall, in plant development and they are also involved in plant-pathogen interaction. PME genes belong to a multigene family in plants, both in dicotyledonous and monocotyledonous species. So far there is a limited knowledge about PMEs in wheat, consequently in this work a genome-wide search was performed on the recently available wheat genome. Forty complete PME sequences and 4 incomplete sequences, 2 of which have been subsequently completed from the bread wheat cv. Bobwhite, were identified. Structural analysis revealed that exon-intron structure is conserved between homoeologous genes sharing a high sequence identity. Expression analysis of PME genes in different bread wheat tissues showed a diverse expression pattern in those tissues and developmental stages analyzed. Most of the members of this gene family underwent a down-regulation following the opening of the florets to perform infection with Fusarium graminearum by using the point inoculation method. Following infection, the expression behavior of specific PME genes was markedly different in the FHB-resistant wheat genotype Sumai3 in comparison to the susceptible cv. Bobwhite, being more strongly induced in the latter than in the former, thus suggesting a possible involvement of PME genes in FHB susceptibility. TRI6 gene of F. graminearum was used to quantify the fungal biomass in three different portions of the spike: the central spikelets directly infected and the upper and lower spikelets respect to the central ones. These analysis showed the presence of the pathogens in the central and upper spikelets and its absence in the lower spikelets. The analysis of PME genes expression in the different parts of the spike showed that the presence of the pathogen in the upper spikelets is associated with an increase of the expression of specific PMEs at 48 hour post infection (hpi). Conversely, in the lower spikelets a down-regulation of PME genes was observed, possibly as result of the stress generated by the opening of the central spikelets for the infection. These preliminary analysis lead us to the identification of those PMEs that could be involved in the susceptibility or resistance in wheat. In order to validate our results and hypothesis, three selected PMEs (PME13, PME21 and PME28) were silenced through RNA interference. Two transgenic lines each silenced for PME13 or PME28 were subjected to a preliminary infection experiment with F. graminearum, but no significant differences were found between the transgenics and the control plants, except between AZ4-11 transgenic line and Svevo plants at 2 days post infection (DPI) .
Silenced PME genes were also searched by a TILLING approach, by screening a durum wheat TILLING population of 4000 plants available at the University of Tuscia. However, the screening of 1920 samples for mutations in the PME13 gene did not identify any silenced genotype. We screened also the recent available TILLING population database developed by the University of California, Davis. We found 28 mutated accessions for 12 different PME genes: STOP codons were found in 18 accessions and splicing sites mutations were found in the remaining 10 accessions.
In conclusion, this genome-wide study of wheat PMEs revealed structural and functional characteristics of this gene family and highlighted the possible involvement of specific PME in wheat-F. graminearum interaction. Silencing of PME13 and PME28 did not modify wheat resistance against F. graminearum in the preliminary infection experiment. However, the selected TILLING mutants for additional PME genes will represent the basis for further study to verify the involvement of PMEs in wheat-pathogen interaction.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
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