Brassinosteroids modulate the efficiency of plant immune responses to microbe-associated molecular patterns

Abstract

Metazoans and plants use pattern recognition receptors (PRRs) to sense conserved microbial-associated molecular patterns (MAMPs) in the extracellular environment. In plants, the bacterial MAMPs flagellin and elongation factor Tu (EF-Tu) activate distinct, phylogenetically related cell surface pattern recognition receptors of the leucine-rich repeat receptor kinase (LRR-RK) family called FLS2 and EF-Tu receptor, respectively. BAK1 is an LRR-RK coreceptor for both FLS2 and EF-Tu receptor. BAK1 is also a coreceptor for the plant brassinosteroid (BR) receptor, the LRR-RK BRI1. Binding of BR to BRI1 primarily promotes cell elongation. Here, we tune the BR pathway response to establish how plant cells can generate functionally different cellular outputs in response to MAMPs and pathogens. We demonstrate that BR can act antagonistically or synergistically with responses to MAMPs. We further show that the synergistic activities of BRs on MAMP responses require BAK1. Our results highlight the importance of plant steroid homeostasis as a critical step in the establishment of plant immunity. We propose that tradeoffs associated with plasticity in the face of infection are layered atop plant steroid developmental programs.

Fig. 1.

BR biosynthesis modulates MAMP signaling. (A) Top: Images representative of Arabidopsis WT Col-0, fls2, and 35S::DWF4. Middle: Microsomal protein extracts prepared from genotypes listed at the top were subjected to anti-BRI1 immunoblot analysis. Bottom: Aniline blue-stained callose deposits in the leaves of the genotypes listed at the top treated with 1 μM flg22. (B) Oxidative burst triggered by 1 μM flg22 in WT Col-0 (blue), fls2 (red), and 35S::DWF4 (yellow) leaf discs measured in relative luminescence units. (C) GUS stains of CYP71A12::GUS line. Seedlings grown in the presence or absence of 5 μM BRZ were left untreated or treated with 1 μM flg22 for 12 h before GUS staining. Washout indicates removal of BRZ during flg22 treatment. Numbers at the bottom indicate the number of roots tested that fall into each category among the 32 roots assayed when BRZ was used in conjunction with flg22. In this assay, 20 roots of 32 displayed a highly attenuated response in the form of small blue spots. (D) Subcellular dynamics of BKI1 upon BL treatment is not affected by flg22 treatments. Subcellular localization of BKI1mCIT is shown in root meristem epidermal cells. BKI1mCIT is localized to the PM and cytosol in the absence of BL treatment (i.e., DMSO) and relocates rapidly from the PM to the cytosol following BL application. Note that BKI1 relocalization to the cytosol after BL treatment is not affected by flg22 treatment even when low concentrations of BL are used (10 nM and 1 nM). This experiment was repeated two times with similar results.

Fig. 2.

BR signaling modulates MTI. (A) Oxidative burst in relative luminescence units, triggered by 1 μM flg22 in leaf discs of the genotypes listed on the top right corner. (B) Aniline blue-stained callose deposits in leaves of the genotypes listed at the bottom treated with 1 μM flg22 or elf18 and 100 μg/mL of PGNs or chitin.

Fig. 3.

Overexpression of BRI1 antagonizes BAK1-mediated MAMP and cell death responses. (A) Top: Aniline blue-stained callose deposits in leaves of the genotypes listed treated with 1 μM flg22. Middle, Bottom: Microsomal protein extracts prepared from the genotypes listed at the top were subjected to an anti-GFP (WB:mCIT) or to an anti-HA (WB:HA) immunoblot analysis to detect the accumulation of mCit or HA tagged proteins. (B) Top: Trypan blue stain of leaves from the genotypes listed at the top. Genotypes of parental and resulting F1 lines are indicated at the top. The control lane (Left) represents F1 plants obtained through a cross between BRI1mCIT and Col-0. Trypan blue stains of F1 plants from the following crosses: BRI1mCit × Col-0 (control; Left), BAK1mCHE × Col-0 (Middle), and BAK1mCHE × BRI1mCit (Right). Two middle panels: Microsomal protein extracts were prepared from the genotypes listed at the top and subjected to anti-GFP and anti-DsRED immunoblot analysis to detect mCitrine and mCherry tagged proteins, respectively. Bottom: Total protein extracts were prepared from the genotypes listed at the top and were subjected to anti-PR1 immunoblot analysis.

Fig. 4.

BR signaling modulates flg22-dependent disease resistance. Growth of PtoDC3000 hrcC was measured in the genetic backgrounds indicated at the bottom of the chart. Four-week-old plants were infiltrated with 10⁵ cfu/mL PtoDC3000 hrcC in the absence (black bars) or presence (yellow bars) of 1 μM flg22. The number of bacteria per area of leaf was determined at 0 and 3 d postinoculation (Experimental Procedures) and plotted on a log10 scale. Values are mean cfu/mL ± 2 SE.

Fig. 5.

Enhanced BR signaling promotes growth of an obligate biotrophic pathogen in a BAK1-dependent manner. (A) Images of representative rosette stage Arabidopsis plants with genotypes listed at the top. Bottom: Quantitative RT-PCR analyses of WRYK6 and WRKY29 transcripts from plants expressing BRI1 (red) or BRI1^sud1 (blue) in WT or bak1 genetic backgrounds. (B) Average fresh-weight ratio of seedlings grown in water (NT) or 1 μM flg22 (T). Genotypes are listed at the bottom. (C and D) Twelve-day-old seedlings of the genetic backgrounds indicated at the bottom of the chart were inoculated with conidiospores of the virulent Hpa isolate Noco2 at 32,000 spores/mL. Sporangiophores were counted 4 d after inoculation on cotyledons (C) and on first true leaves (D) for each of the indicated genetic backgrounds. Means, sample sizes, and 2 × SE are presented in Table S1. The experiment was repeated twice. sp, sporangiophores per cotyledon (C) or sporangiophores per leaf (D).