3. Results
3.4 Reverse genetic analysis of anac071
3.5.4 Analysis of the role of At5g24080 in defence against Pseudomonas syringae, Botrytis cinerea and
Expression of the G-type LecRLK gene At5g24080 is strongly up-regulated during infection with chlorosis-inducing isolates of the vascular plant pathogen Verticillium. In addition, Arabidopsis G-type LecRLK LORE and lectin receptor kinases from various plant species were shown to function in defence against bacterial as well as fungal pathogens and herbivorous insects (Kimet al., 2009; Chenet al., 2006; Gilardoniet al., 2011; Chenget al., 2013; Cole and Diener, 2013; Liu et al., 2015; Ranfet al., 2015), suggesting that AT5G24080 as a LecRLK may be involved in plant immunity. Sinceat5g24080 mutants did not show any altered disease phenotypes in response to Verticillium, next susceptibility ofat5g24080-1, at5g24080-2 and at5g24080-3 mutants to a biotrophic, necrotrophic and a hemi-biotrophic phytopathogen was analysed. The set of tested pathogens included the hemibiotrophic bacterium Pseudomonas syringae, the necrotrophic fungus Botrytis cinerea and the obligate biotrophic oomycete Hyaloperonospora arabidopsidis.
Publically available expression data indicate, that At5g24080 is induced 2, 6 and 24 h after infection of 5-week-old A. thaliana Col-0 with the virulentPseudomonas syringae pv. tomato DC3000 (Pst DC3000) as well as 6 and 24 h after infection with the avirulent Pseudomonas syringae pv. tomato avrRpm1 as compared to mock treatment (Arabidopsis eFP Browser, Winter et al., 2007). In addition, expression of the At5g24080 gene is up-regulated 4 h after infiltration with the epitope of bacterial flagellin, flg22 and bacterial hairpin protein HrpZ, which both represent well known elicitors of plant defence. For these reasons, at5g24080 mutant susceptibility to the hemibiotrophic bacteriumPseudomonas syringaewas assessed.
a wild-type control andeds1-2 as a susceptible control. EDS1 represents a positive regulator of basal and TNL-type R-protein mediated resistance (Wiermer et al., 2005). Bacterial titre at day 0 did not significantly differ in all tested genotypes, indicating a uniform infiltration (Fig. 17). At day 3 Pst DC3000 ΔAvrPto/AvrPtoB proliferation was not significantly altered in at5g24080 mutants as compared to Col-0 wild-type (Fig. 17), suggesting that at5g24080 is not implicated in disease resistance to this bacterial pathogen.
Next, susceptibility of at5g24080 to the necrotrophic fungal pathogen Botrytis cinerea was analysed. Leaves of 5-week-old at5g24080-1 mutants were drop inoculated with B. cinerea isolate B05.10 spore suspension and the diameter of necrotic lesion measured at 3 dpi.
A. thaliana Col-0 was used as a wild-type control andmpk3 DG as well asmos7-1as susceptible controls. MPK3 is a component of a MAP kinase cascade which leads to induction of pathogenesis related genes, whereas MOS7 represents a nucleoporin protein which is required
d0 * syringae pv. tomato (Pst) strain DC3000 (ΔAvrPto/AvrPtoB) growth in Arabidopsis Col-0 wild-type, at5g24080 and eds1-2 leaves.
eds1-2 represents a susceptible control. 5- to 6-week-old plants were vacuum infiltrated with a bacterial suspension of 1 · 105colony forming units (c.f.u.) per ml. Leaf samples were taken in duplicates at day 0 as a control for equal infiltration (grey bars). Day 3 leaf samples were taken in triplicates (black bars). Error bars represent standard deviation between replicates.
*P < 0.05 using Student’s t-test for pairwise comparison of wild-type and mutant. The experiment was repeated twice with similar results.
al., 2010; Maoet al., 2011). Both proteins were shown to be required for defence responses to B. cinerea (Renet al., 2008; Genenncheret al., 2017). The mean diameter of necrotic lesions onat5g24080-1 leaves was not significantly altered as compared to Col-0 wild-type (Fig. 18), suggesting that at5g24080-1 does not show enhanced susceptibility to B. cinerea. As a consequence, AT5G24080 is not likely involved in disease resistance to this fungal phytopathogen.
Subsequently, susceptibility of at5g24080 to the obligate biotroph Hyaloperonospora arabidopsidis was assessed. 2-week-old at5g24080-1, at5g24080-2 and at5g24080-3 were spray inoculated with a spore suspension of theH. arabidopsidis isolate NOCO2 and oomycete proliferation was quantified at 6 dpi. NOCO2 represents an A. thaliana Col-0 adapted isolate of H. arabidopsidis. For this reason A. thaliana Col-0 was used as a wild-type control.
Furthermore, snc1 was used as a resistant control whereas eds1-2 represented a susceptible control. Thesnc1mutant carries a gain of function mutation in a TNL-type R-protein resulting in a constitutive immune response (Liet al., 2001; Zhanget al., 2003).
Figure 18. Disease susceptibility of Arabidopsis Col-0 wild-type, at5g24080-1, mpk3 DG and mos7-1 to Botrytis cinerea B05.10. mpk3 DG and mos7-1 represent susceptible controls. Leaves of 5-week-old plants were inoculated with 6 µl droplets of B. cinerea spore suspension at a concentration of 5 · 104spores per ml. Necrotic lesion diameter was measured 3 days post inoculation (dpi) with a digital calliper. Error bars represent standard deviation between 30-45 measurements. *P < 0.05 using Student’s t-test for pairwise comparison of wild-type and mutant. The experiment was repeated twice with similar results.
susceptible to NOCO2 infection as compared to the susceptible eds1-2 control, from which 30·105 NOCO2 spores per ml were recovered at 6 dpi (Fig. 19). In addition to the experiment presented in this thesis, two further NOCO2 infection experiments were performed. In one of the two repetitions, all testedat5g24080 mutants were more susceptible to NOCO2 as compared to Col-0 wild-type, whereas susceptibility ofat5g24080 mutants was comparable to wild-type in the second repetition.
As a conclusion, it can be stated that at5g24080-1, at5g24080-2 and at5g24080-3 mutations did not result in enhanced susceptibility to the hemibiotrophic bacterial pathogenPseudomonas syringae and necrotrophic fungusB. cinerea. However, at5g24080 mutants showed a trend to enhanced susceptibility toH. arabidopsidis isolate NOCO2. Yet, infection experiments have to be repeated in the future.
Figure 19. Hyaloperonospora arabidopsidis isolate NOCO2 proliferation 6 days post infection of Arabidopsis Col-0 wild-type, at5g24080, snc1 and eds1-2 mutant. snc1 represents a resistant control, whereaseds1-2 is a susceptible control. 35-45 2-week-old seedlings were spray inoculated with a suspension of 5 · 104 spores per ml. Error bars represent standard error between n = 4 replicates. *P < 0.05 using Student’s t-test for pairwise comparison of wild-type and mutant. The experiment was performed three times. Similar results were observed in 2 out of 3 experiments.