Type-ll Metacaspases Mediate the processing of Plant Elicitor Peptides in Arabidopsis

Plants can produce animal cytokine-like immune peptides,among which plant elicitor peptides(Peps)derive from the C termini of their precursors(PROPEPs).Recently,the functions of Peps have been expanded beyond plant immunity.However,a long-standing enigma is how PROPEPs are processed into Peps.Here,we report that the Ca2+-dependent type-ll metacaspases(MCs)constitute the proteolytic enzymes to mediate PROPEP processing in Arabidopsis.In protoplasts,co-expression of PROPEP1 with type-ll MCs,including MC4 to MC9,can promote the generation of processed Pep1.Destruction of the catalytic cysteine residue in MC4 or the conserved arginine residue preceding the Pep1 sequence blocks PROPEP1 cleavage,whereas the bacterial elicitor flg22 enhances the MC4-mediated PROPEP1 processing.MC4 cleaves PROPEP1 in vitro and also cleaves PROPEP2 to PROPEP8,but,surprisingly,not PROPEP6 in protoplasts.Domain swapping between PROPEP1 and PROPEP6 suggests a hidden role of the sequence context upstream of the Pep sequence for PROPEP processing.flg22-induced PROPEP1 processing and B otrytis cinerea resistance are severely impaired in the m c4/5/6/7 quadruple-mutant plants.Taken together,our study identifies the type-ll MCs as new players in Pep signaling,and lays the foundation for understanding the regulation of multifaceted functions of Peps in plant immunity and beyond.

Engineering plants to secrete affinity-tagged pathogen elicitors for deciphering immune receptor complex or inducing enhanced immunity

Plant cells mount plenty of pattern-recognition receptors(PRRs)to detect the microbe-associated molecular patterns(MAMPs)from potential microbial pathogens.MAMPs are overrepresented by proteinaneous patterns,such as the flg22 peptide from bacterial flagellin.Identification of PRR receptor complex components by forward or reverse genetics can be time/labor-consuming,and be confounded by functional redundancies.Here,we present a strategy for identifying PRR complex components by engineering plants to inducibly secrete affinity-tagged proteinaneous MAMPs to the apoplast.The PRR protein complexes bound to self-secreted MAMPs are enriched through affinity purification and dissected by mass spectrometry.As a proof of principle,we could capture the flg22 receptor FLS2 and co-receptor BAK1 using Arabidopsis plants secreting FLAG-tagged flg22 under estradiolinduction.Moreover,we identified receptor-like kinases LIK1 and PEPR1/PEPR2 as potential components in the FLS2 receptor complex,which were further validated by protein–protein interaction assays and the reverse genetics approach.Our study showcases a simple way to biochemically identify endogenous PRR complex components without overexpressing the PRR or using chemical crosslinkers,and suggests a possible crosstalk between different immune receptors in plants.A modest dose of estradiol can also be applied to inducing enhanced immunity in engineered plants to both bacterial and fungal pathogens.