The role for the exocyst complex subunits Exo70B2 and Exo70H1 in the plant-pathogen interaction

Abstract

Recently, the octameric vesicle-tethering complex exocyst was found in plants and its importance for Arabidopsis morphogenesis was demonstrated. Exo70 exocyst subunits in plants, unlike in yeasts and mammals, are represented by a multigene family, comprising 23 members in Arabidopsis. For Exo70B2 and Exo70H1 paralogues, transcriptional up-regulation was confirmed on treatment with an elicitor peptide, elf18, derived from the bacterial elongation factor. Their ability to participate in the exocyst complex formation was inferred by the interaction of both the Exo70s with several other exocyst subunits using the yeast two-hybrid system. Arabidopsis plants mutated in these two genes were used to analyse their local reaction upon inoculation with Pseudomonas syringae pv. maculicola and the fungal pathogen Blumeria graminis f. sp. hordei. The Pseudomonas sensitivity test revealed enhanced susceptibility for the two exo70B2 and one H1 mutant lines. After Blumeria inoculation, an increase in the proportion of abnormal papilla formation, with an unusual wide halo made of vesicle-like structures, was found in exo70B2 mutants. Intracellular localization of both Exo70 proteins was studied following a GFP fusion assay and Agrobacterium-mediated transient expression of the constructs in Nicotiana benthamiana leaf epidermis. GFP-Exo70H1 localizes in the vesicle-like structures, while GFP-Exo70B2 is localized mainly in the cytoplasm. It is concluded that both Exo70B2 and Exo70H1 are involved in the response to pathogens, with Exo70B2 having a more important role in cell wall apposition formation related to plant defence.

Fig. 1.

The RT-PCR analysis of nine different Arabidopsis Exo70 mRNAs after treatment with elicitor peptide elf18 (c, control; e, elf18 treatment; M, marker).

Fig. 2.

The position of T-DNA insertions in three Arabidopsis mutant lines. (A) SALK_091877 (exo70B2-1), SAIL_339-D07 (exo70B2-2), and SALK_042456 (exo70H1). The RT-PCR (B) confirms that these mutants are knock-out lines, since no DNA fragments were obtained by RT-PCR (with the exception of a weak band in exo70B2-1; lanes 1–4,WT, exo70B2-2, exo70B2-1, exo70H1, respectively, with Exo70B2 primers; lanes 5–8, the same arrangement of cDNAs was used to amplify the Exo70H1 fragment).

Fig. 3.

Two hybrid assay in S. cerevisiae: interactions of Exo70B2 and Exo70H1 with several other exocyst subunits and SNARE protein AtSNAP33.

Fig. 4.

Test of susceptibility to Pseudomonas of three exocyst mutant lines: two exo70B2s and exo70H1 compared to WT and sensitive mutant npr1. Relative number of P. syringae pv. maculicola colonies out of inoculated leaves for five different lines are presented as ×10⁵ colony forming units per cm² (cfu cm⁻²).

Fig. 5.

Vesicular-like papillae distribution and penetration of B. graminis f. sp. hordei in WT, exo70B2-1, exo70B2-2, and exo70H1 mutants. Abnormal papillae with vesicular halo and successful penetration of fungi (either as an elongated penetration peg or the presence of haustorium) are counted. For each genotype, 50–100 spores from 4–5 leaves were inspected.

Fig. 6.

(A) Papilla structure in WT and in mutant lines. The fine papilla structure for each of the genotypes was observed by confocal microscopy combined with the bright field. The whole spore with cytoplasm surrounding are shown in the upper row (bar=20 μm); the lower row shows in more details papilla structures (bar=5 μm). The vesicular halo is visible in exo70B2-1and exo70B2-2. (B) The diameters of papillae and the diameters of the area of papillae with vesicular halos were measured for four different genotypes. The vesicular halo is significantly bigger for papillae of exo70B2 mutants.

Fig. 7.

The localization of N-GFP tagged Exo70B2 and Exo70H1 transiently expressed in N. benthamiana epidermal cells after agroinfiltration. Typical intracellular localization of GFP-Exo70B2 (A–C) and GFP-Exo70H1 (D–E) observed 24–36 h post-infiltration is shown. Details of peri-nuclear and intra-nuclear localization for Exo70B2 (C) and Exo70H1 (F) are shown on the right. (A, D) Bars 20 μm; (B, C, E, F) bars 10 μm).