Arabidopsis PEN2, a promising gene in upraising penetration resistance against rice necrotrophic fungus Rhizoctonia solani

Rhizoctonia solani, a soilborne necrotroph, causes sheath blight in rice which poses a major threat to global rice production. Besides rice sheath blight, it has a wide host range of other economically important crops like soybean, sugarcane, maize etc. Despite being the most hostile fungus, the mechanism involved in the R. solani pathobiology is poorly understood. Non-host resistance (NHR) is an emerging concept that allows breeders to transfer traits to food crops that would impart a broad-spectrum disease resistance. Several NHR genes are known to function against different pathogens of which Arabidopsis PEN1, PEN2 and PEN3 have been reported to limit the entry of non-adapted powdery mildews and provide cell wall based defenses against different fungi. Till now, there has been no study regarding the involvement of these PEN genes against R. solani. In this study, we have screened pen1, pen2-3 and pen3-1 against R. solani to explore their contribution in penetration resistance. Among the three pen mutants studied, pen2-3 allowed maximum penetration during the early hours of infection. R. solani colonization was also observed in pen1 and pen3-1 but the effect was less drastic than pen2-3, suggesting the involvement of PEN2 in pre-invasive defense. To validate our hypothesis, we screened a complemented pen2 accession, PEN2-GFP, which showed restored penetration resistance comparable to Col-0. Altogether, our results demonstrate that PEN2 is involved in pre-penetration resistance, and contributes to NHR by enhanced disease resistance to R. solani.

106 acid: water -1:1:1:1, v/v). It was further destained with lactophenol, mounted onto glass slide 107 with 50% glycerol and examined under bright field microscope. For DAB staining, infected 108 leaves were incubated in 1mg/mL aqueous DAB solution for 8h in dark following which stain 109 was replaced with water and incubated in similar conditions. Destaining was performed with 110 acetic acid: ethanol (96:4 v/v) and mounted with 50% glycerol and visualized under brightfield 111 microscope. For aniline blue staining, leaves were cleared in alcoholic lactophenol and stained 112 overnight with 0.01% aniline blue in 150mM K 2 HPO 4 , pH 9.5. stained leaves were equilibrated 113 with 50% glycerol and observed under UV excitation. 114 Extraction and Estimation of Chlorophyll content 115 One gram of treated and untreated leaves were taken at 30 and 48hpi and ground with 20 mL of 116 80% acetone. It was then centrifuged at 5000 rpm for 5 minutes. The supernatant was collected 117 in a tube and the process was repeated until the residue appears colorless. Supernatant was made 118 upto 100 mL with 80% acetone. The absorbance of the solution was recorded at 645 nm and 663 119 nm against the blank solvent (acetone) (Rajalakshmi & Banu 2013). The concentrations of 120 chlorophyll a, chlorophyll b and total chlorophyll was measured as mg/g of the sample and were    187 Deposition of H 2 O 2 was detected as dark yellowish-brown precipitate (Fig. 2). As expected, 188 pen2-3 exhibited relatively higher level of accumulation of H 2 O 2 at more sites of infection, 189 followed by pen1 and pen3-1 as compared to Col-which can be correlated with increased cell 190 death rate (Fig. 2). Callose deposition was examined using aniline blue stain. The leaves of wild 191 type showed reduced callose deposition and mutant plants accumulated significantly more 192 localized callose (Fig. 3). Significantly higher amount of callose deposition was found in the 193 leaves of infected pen2-3 among all indicating compromised disease resistance in pen2-3, 194 substantiating the microscopic data.
218 formation at 30hpi and 40hpi in response to R. solani (Fig. 5). This experiment corroborated our 219 hypothesis that PEN2 enhance penetration resistance against R. solani.    Each experiment was carried out with three biological replicates. Scale bar-50µm.

Figure 3
Accumulation of callose at sites of infection in Arabidopsis leaves Leaves of wild type and pen mutants of Arabidopsis were stained with aniline blue after 30hpi and 48hpi. Callose deposition was observed UV excitation of a fluorescence microscope.

Figure 4
Macroscopic quantification of disease progression in Arabidopsis wild type and pen mutants Four week old plants were infected with R. solani sclerotia and photographed at 0dpi, 1dpi, 2dpi and 3dpi. Leaves inoculated with water were used as control. The coverage of necrotic lesions increased with time in each accession with pen2-3 being the most affected. The experiment was carried out three times, and each contained three biological replicates.

Figure 5
Comparison of infection structure of PEN2-GFP and pen2-3 as compared to wild type Col-0 Leaves of wild type Col-0, PEN2-GFP and pen2-3 mutants of Arabidopsis were stained with trypan blue after 30hpi and 48hpi. Maximum cushions formed at 48hpi in pen2-3. Infection cushions are indicated by arrows. Scale bar ~ 50µm.