PCR based confirmation of homozygous PENETRATION mutants used in the study
(a) the T-DNA insertion mutant of pen1 (SALK_004484C) was confirmed using a T-DNA left border primer indicating the presence of insert in the mutant. (b) pen2-3 was confirmed using a CAPS marker indicating the presence of an additional restriction site for BsmAI restriction enzyme. (c) pen3-1 was also confirmed using a CAPS marker showing deletion of HphI restriction site in the mutant.
Infection phenotypes of Arabidopsis wild type and pen mutants upon infection with R. solani
Leaves of Col-0 and pen mutants were stained with trypan blue after 6, 12, 24, 30, 36 and 48hpi to observe the extent of disease progression. Stained leaves were observed under bright field microscope. Leaves inoculated with water were used as control. Scale bar = 100µm
Total chlorophyll estimation upon infection with R. solani on Arabidopsis wild type (Col-0) and pen mutants
Total chlorophyll content measured in leaves of wild type and pen mutants of A. thaliana after 30 and 48hpi with R. solani sclerotia. Leaves treated with water served as control. Data represent the mean ± SEM of three biological replicates (n = 3). The experiment was carried out three times with similar results
SEM micrographs of Arabidopsis pen mutants showing post infection hyphal colonization by R. solani at 30hpi and 48hpi
Leaves of Arabidopsis wild type (Col-0) and pen mutants inoculated with water as control. Col-0 and pen mutants inoculated with R. solani sclerotia showed disease progression with profuse hyphal branching after 30hpi. pen2-3 showed sporadic onset of infection cushion formation (ioc; dashed arrow) after 30hpi. Col-0 and pen mutants inoculated with R. solani sclerotia observed after 48hpi. Dense infection cushions (ic; solid arrows) at 48hpi in pen2-3. Swollen hyphal tip formed lobate apperesoria (la) at 48hpi in pen3-1. Scale bar ~ 50µm
Macroscopic quantification of disease severity of R. solani infected Arabidopsis pen mutants
Macroscopic necrotic lesions were quantified in terms of area using ImageJ software after 1, 2 and 3dpi. Data represent percentage mean area ± SEM (n=5).