Regulation of plant immunity to necrotrophic fungi
We examined Arabidopsis resistance mechanisms to Botrytis cinerea and Alternaria brassicicola, two necrotrophic fungal pathogens with overlapping pathogenesis strategies. B. cinerea is the causal agent of gray mold whereas A. brassicicola causes the black spot disease in cruciferous plants.
In an effort to isolate regulators of plant defense, we used functional genomics approaches to identify genes involved in immune responses to necrotrophic fungi. Genome-wide expression profiling of B. cinerea inoculated plants and functional analysis using T-DNA insertion alleles of induced genes identified UPI (UNIQUE SERINE PROTEASE INHIBITOR and PGN (PENTATRICOPEPTIDE REPEAT PROTEIN FOR GERMINATION ON NaCl) as important components of defense against B. cinerea and A. brassicicola. The UPI gene is significantly induced by oxidative stress, the hypersensitive response, wounding, and pathogen infection dependent on functional jasmonate (JA) responses. Ethylene (ET) suppresses UPI gene expression as evidenced by abrogation of UPI expression following treatment with ACC, the ET precursor, and its increased levels in mutants impaired in ET responses. Thus, JA and ET function antagonistically in the regulation of UPI gene expression during plant defense, contradicting their normally synergistic interaction during necrotrophic infection. Overall, our data suggest UPI is a JA regulated defense molecule required for resistance to necrotrophic fungi and the regulation of flowering time.
In parallel, we studied the function of PGN encoding a member of the Arabidopsis pentatricopeptide repeat (PPR) protein family. Generally, PPR proteins are known to function in RNA metabolism including transcript editing, splicing, and stability as well as the control of translation. The pgn mutant is susceptible to B. cinerea and A. brassicicola but shows wild type resistance to Pseudomonas syringae. In addition, the pgn mutant is hypersensitive to abscisic acid (ABA), glucose and salinity suggesting that PGN mediates biotic and abiotic stress tolerance. Inhibition of ABA synthesis and responses partially alleviates pgn sensitivity to glucose suggesting the mutant has increased ABA accumulation. This is consistent with the increased induction of NCED3, a regulator of stress induced ABA biosynthesis, in pgn following treatment with salt and ABA.
We also analyzed the genetic interaction between BOTRYTIS-INDUCED KINASE1 (BIK1) and key Arabidopsis defense regulatory genes. The genetic interactions between BIK1 and regulators of salicylic acid (SA), JA, ET and HISTONE MONOUBIQUITINATION1 (HUB1) dependent defense responses were determined using double mutants. BIK1 mediates distinct responses to biotrophic (P. syringae) and necrotrophic pathogens, providing an avenue for the genetic analysis of the complex regulatory networks of Arabidopsis defense. Removal of SA through genetic crosses to sid2 (impaired in SA synthesis), pad4 (impaired in SA accumulation), or nahG (impaired in SA accumulation) plants restores wild-type resistance to necrotrophic infection suggesting increased SA promotes bik1 susceptibility. NPR1, the central regulator of SA signaling and SA dependent defense responses, contributes to limiting pathogen growth and disease symptoms as revealed from the increased susceptibility of bik1npr1 to necrotrophic infection relative to both parental mutants. Interestingly, bik1 susceptibility is independent of the reduced PDF1.2 expression, a plant defensin gene used as a molecular marker of ET and JA dependent defenses generally associated with resistance to necrotrophs.
Despite susceptibility to necrotrophic fungi, bik1 is resistant to the bacterial pathogen P. syringae pv. tomato (Pst). The addition of pad4 or sid2 mutation partially restored bik1 susceptibility to Pst whereas npr1 and coi1 had no effect. These results are similar to those obtained following necrotrophic infection, with resistance largely dependent on SA accumulation and PAD4 responses. However, bik1pad4 and bik1sid2 retained significant resistance relative to sid2 and pad4, respectively, consistent with the idea of BIK1 function in SA-dependent and independent pathways. (Abstract shortened by UMI.)