3. Results
3.4 Reverse genetic analysis of anac071
3.4.2 Analysis of anac071 disease phenotype during Verticillium chlorosis and wilting isolate infection 64
In order to analyse the potential role of ANAC071 in the A. thaliana – Verticillium chlorosis isolate interaction, macroscopic disease symptoms ofanac071-1 andanac071-2 mutants were analysed duringVerticillium challenge. Macroscopic disease symptoms of the anac071-1 and anac071-2 mutant were not altered during Verticillium chlorosis-inducing isolate c-VL43 and c-V76 as well as wilting isolate w-JR2 infection as compared to wild-type (Fig. 8). As Col-0 wild-type, both testedanac071 mutants showed chlorosis and early senescence of older rosette leaves during infection with the V. longisporum chlorosis isolate c-VL43 and V. dahliae chlorosis isolate c-V76 (Fig. 8A, middle panels). In addition, comparable with Col-0 wild-type, bothanac071 mutants demonstrated wilting symptoms on older rosette leaves duringV. dahliae wilting isolate w-JR2 infection (Fig. 8A, right panel).
Stunting of the rosette was quantified as an indicator for severity of disease symptoms. Leaf area of mock treated controls was set to 100 % and leaf area of infected plants calculated as percentage of mock control. A pairwise comparison of wild-type and mutant was carried out using t-test, to analyse statistical significance. Leaf area ofanac071-1 andanac071-2 mutants did not significantly differ from Col-0 wild-type duringVerticillium chlorosis isolate c-VL43, c-V76 and wilting isolate w-JR2 infection (Fig. 8B), implying that mutation of the ANAC071 gene does not affect severity ofVerticillium induced disease symptoms.
Figure 8. Disease phenotypes of A. thaliana Col-0 wild-type, anac071-1 and anac071-2 during infection with V. longisporum isolate c-VL43 as well as V. dahliae isolates c-V76 and w-JR2 (A) Disease symptoms at 21 days post infection (dpi). Insets represent magnifications of areas marked with dotted boxes. Arrowheads indicate wilting leaves. (B) Leaf area measurement at 21 days post infection. Leaf area of mock treated controls was set to 100 % and leaf area of infected plants calculated as percentage of mock control. Error bars represent standard deviation between n = 4 replicates.
Statistical significance was tested using Student’s t-test for pairwise comparison of wild-type and mutant. (n.s.) not significant. The experiment was performed once.
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transdifferentiation in A. thaliana Col-0 wild-type and anac071-1 as well as anac071-2 leaf vascular bundles were performed during Verticillium chlorosis isolate infection. Detached leaves were fed with the water soluble dye safranin-O, in order to visualise vascular tissue. In the mock treated Col-0 wild-type, vascular bundles are surrounded by chloroplast containing bundle sheath cells (Fig. 9A). During chlorosis isolate c-VL43 and c-V76 infection bundle sheath cells transdifferentiate to xylem elements, showing characteristic annular, helical and reticulate secondary cell wall fortifications (Fig. 9B and C). In contrast, during wilting isolate w-JR2 infection bundle sheath cell layer does not transdifferentiate (Fig. 9D). Like Col-0 wild-type, leaf vascular bundles of mock treated and wilting isolate w-JR2 infected anac071-1 andanac071-2 did not exhibit developmental changes (Fig. 9E, H, I and L). Furthermore, both tested anac071 mutants showed wild-type like bundle sheath cell transdifferentiation into xylem elements during chlorosis isolate c-VL43 and c-V76 infection (Fig. 9F, G, J and K), indicating thatANA071 does not play a role in bundle sheath cell transdifferentiation.
ANAC071 is involved in tissue reunion of incised Arabidopsis inflorescence stems. Tissue reunion is incomplete in theanac071-1 mutant andANAC071gene-suppressing transformants (Asahinaet al., 2011; Pitaksaringkarnet al., 2014). For this reason, it is conceivable to postulate that defects inANAC071 may have an effect on connectivity ofde novo formed xylem elements.
The water soluble safranin-O dye is transported with the transpiration stream within the leaf vessel elements (Freeman and Beattie, 2009). Thus, safranin-O staining allows to test connectivity and functionality of the de novo formed xylem elements in water transport.
Safranin-O was detectable in the lumen of newly formed xylem cells in Col-0 wild-type but also in anac071 mutants (Fig. 9B, C, F, G, J and K), indicating that de novo formed xylem elements were connected to the vascular system and functional in water transport.
Taken together, neither theanac071-1 nor the anac071-2 mutant showed altered macroscopic disease symptoms or significant differences in disease symptom severity during infection with the V. longisporum chlorosis isolate c-VL43 and V. dahliae chlorosis isolate c-V76 as well as V. dahliae wilting isolate w-JR2 as compared to Col-0 wild-type. Moreover, both tested anac071 mutants showed wild-type like bundle sheath cell transdifferentiation into connected, functional xylem elements during chlorosis isolate c-VL43 and c-V76 infection. In conclusion, these results suggest that ANAC071 does not play a role in establishment of chlorosis disease symptoms and bundle sheath cell transdifferentiation during Verticillium chlorosis isolate infection.
Figure 9. Analysis of bundle sheath cell transdifferentiation into functional tracheary elements in leaf vascular bundles of A. thaliana Col-0 wild-type and anac071 mutants 21 days post infection (dpi) with V. longisporum isolate c-VL43 as well as V. dahliae isolates c-V76 and w-JR2.Detached leaves were fed with the water-soluble dye safranin-O. Figures(A-L) show bright field images of leaf vascular bundles with the focal plane set to the xylem cell lumen to demonstrate staining of xylem sap. Insets represent magnifications of areas marked with dotted boxes. Asterisks indicate chloroplast containing bundle sheath cells, whereas arrowheads point
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3.5 Reverse genetic analysis of at5g24080
RNA-sequencing analysis performed in this study identified aNicotiana benthamianahomolog of A. thaliana protein kinase At5g24080 as a V. dahliae chlorosis isolate induced gene.
At5g24080was selected as a candidate gene for detailed analyses, because it is significantly as well as highly induced by chlorosis isolate infection and no molecular analyses of At5g24080 were published so far (November 2017). Semi-quantitative RT-PCR and qPCR revealed that A. thaliana At5g24080was specifically up-regulated during infection with theV. longisporum chlorosis-inducing reference isolate c-VL43, as well as the V. dahliae chlorosis-inducing reference isolate c-V76 but not the wilting-inducing reference isolate w-JR2 (Fig. 4 and 5). As a protein kinase, it may play a potential role in signal transduction required for the establishment of the chlorosis phenotype.