Multi-transgene-stacking of glycosidase inhibitor genes to improve resistance against fungal pathogens in wheat
Author(s)
Kalunke, Raviraj Mahadeo
Date Issued
April 11, 2012
Type
Doctoral Thesis
Abstract
The diseases caused by pathogenic fungi are major problem for grain production. It reduces the
productivity and causes contamination of grain with mycotoxins, compounds that are harmful to
human health and animals. Disease control can be done by chemical treatments but these are costly
and adverse affect on the environment. The most efficient strategy to control crop diseases is the
development of resistant genotypes. This goal can be achieved by enhancing specific plant defence
mechanisms, among which the reinforcement of plant cell wall compartment can be one of the most
efficient because most pathogens have to overcome this barrier to colonize the host tissue. Plant cell
walls consist mainly of polysaccharides (i.e. cellulose, hemicelluloses and pectins) and play an
important role in defending against pathogens. Many pathogenic fungi can produce a range of cell
wall-degrading extracellular enzymes (CWDEs) that are capable of depolymerizing the
polysaccharides in the host cell wall. CWDEs are the polysaccharide degrading enzymes, including
exo- and endo-polygalacturonases, pectin methylesterases, pectin and pectate lyases, acetyl
esterases, xylanases and a variety of endoglucanases that cleave cellulose, xyloglucan and other
glucans. During the defence response plant produces protein inhibitors of CWDE. The
polygalacturonase inhibitor protein (PGIP) inhibits endopolygalacturonase (PG) secreted by fungal
pathogens. The degree and pattern of cell wall pectin methyl-esterification can also influence plant
resistance, as highly methyl-esterified pectin is less susceptible to the hydrolysis by fungal PGs.
Pectin methyl esterification is controlled by the interaction between pectin methyl esterase (PME)
and its protein inhibitor (PMEI). The PGIP and PMEI play an important role in preventing action of
PG on pectin. Cereals contain Xylanase inhibitors (XIs) which inhibit microbial Xylanases, from
glycoside hydrolase families 10 and 11. Endo β-1,4-xylanases (xylanases; EC 3.2.1.8) are key
enzymes in the degradation of arabinoxylans (AXs), the main non-starch polysaccharides from
cereal cell walls.
The main objective of this study was to pyramid glycosidase inhibitor genes (PGIP, PMEI and XI),
evaluate their co-segregation frequency and their combined effect on wheat resistance against
fungal pathogens in wheat. In order to achieve this goal, multi-transgenes-stacking of PGIP, PMEI
and XI in wheat was carried out by two approaches, multiple plasmid co-transformation and
classical crossing.
In the co-transformation approach, four plasmids containing Pvpgip, Acpmei, Taxi-III and bar genes
in wheat T. durum cv. Svevo were co-transformed by particle bombardment. Total sixteen T0 plants
were obtained, among them seven plants showed all four genes which represents 58% cotransformation
frequency for four transgenes. Further, seeds were harvested and used for T1
segregation analysis. All the four transgenes co-segregated at ratio of 3:1, indicating that all four
genes are tightly linked in all seven lines. Similar co-transformation experiments were performed in
bread wheat (T. avesticum cv. Bobwhite) using four plasmids each containing PvPGIP2, AcPMEI
Xip-III and bar genes. Total eighteen T0 plants were obtained, among them five plants showed all
four transgenes which represent 27.77 % co-transformation frequency for four transgenes. Further,
seeds were harvested and used for T1 segregation analysis. All the four transgenes co-segregated at
ratio of 3:1, indicating that all four genes tightly linked in all lines.
Segregation analysis in both durum and bread wheat showed about 70% progeny contained all
three transgenes of interest together and also tightly linked.
In crossing approach, parental lines of durum wheat cv Svevo each expressing single glycosidase
inhibitor were crossed. First, plant containing PvPGIP2/AcPMEI were obtained, and these plants
were crossed with a line expressing TAXI-III. In F1 progeny, a total of nineteen F1 plants were
obtained, with only two plants possessing all three transgenes. Among these two plants, one plant
(M011-1-5) exhibited high level of expression for PGIP and TAXI-III along with inhibition activity
but, for PMEI no inhibition activity was observed, probably caused by silencing events. The
remaining one plant (M011-1-4) exhibited high level expression for all three genes and their
corresponding inhibitory activity. The segregation analysis F2 progeny of this plant (M011-1-4)
showed only 10% of the segregating progeny with all three transgenes of interest
Our results indicate that multi-stacking-transgenes using co-transformation can perform better than
the classical crossing approach since more than 70% of the progenies were with three transgenes of
interest and also tightly linked. On the contrary, in the crossing method only 10% of the segregating
progeny contained all three glycosidase inhibitors.
Three transgenic durum wheat lines containing tightly linked PvPGIP2, AcPMEI and TAXI-III
(lines MJ56-5, MJ56-16a and MJ56-16b) were analyzed for transgene expression and inhibition
activity. All three transgenic lines exhibited high level of expression for PMEI and TAXI-III along
with inhibition activity but, for PGIP a very low expression and no inhibition activity was observed,
probably becaused of gene silencing events. T2 generations of these three lines were used to analyze
their resistance response to B. sorokiniana infection. The results showed about 70 % reduction in
Leaf bloch disease symptom. This level of protection is higher than that obtained with the
overexpression of only AcPMEI (about 55%) in durum wheat (Volpi et al. 2011), probably because
of the co-presence of TAXI-III. However, the possibility that a higher level of PMEI activity in
these transgenic plants, compared to those analysed by Volpi et al. (2011) is responsible for the
higher protection cannot be ruled out.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
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