Plant elicitor peptide signalling confers rice resistance to piercing-sucking insect herbivores and pathogens.

Rice is a staple food crop worldwide, and its production is severely threatened by phloem-feeding insect herbivores, particularly the brown planthopper (BPH, Nilaparvata lugens), and destructive pathogens. Despite the identification of many BPH resistance genes, the molecular basis of rice resistance to BPH remains largely unclear. Here, we report that the plant elicitor peptide (Pep) signalling confers rice resistance to BPH. Both rice PEP RECEPTORs (PEPRs) and PRECURSORs of PEP (PROPEPs), particularly OsPROPEP3, were transcriptionally induced in leaf sheaths upon BPH infestation. Knockout of OsPEPRs impaired rice resistance to BPH, whereas exogenous application of OsPep3 improved the resistance. Hormone measurement and co-profiling of transcriptomics and metabolomics in OsPep3-treated rice leaf sheaths suggested potential contributions of jasmonic acid biosynthesis, lipid metabolism and phenylpropanoid metabolism to OsPep3-induced rice immunity. Moreover, OsPep3 elicitation also strengthened rice resistance to the fungal pathogen Magnaporthe oryzae and bacterial pathogen Xanthamonas oryzae pv. oryzae and provoked immune responses in wheat. Collectively, this work demonstrates a previously unappreciated importance of the Pep signalling in plants for combating piercing-sucking insect herbivores and promises exogenous application of OsPep3 as an eco-friendly immune stimulator in agriculture for crop protection against a broad spectrum of insect pests and pathogens.

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 estradiol induction. 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 cross-linkers, 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.

Hidden prevalence of deletion-inversion bi-alleles in CRISPR-mediated deletions of tandemly arrayed genes in plants.

Tandemly arrayed genes (TAGs) with functional redundancy and chromosomal linkage constitute 14 ~ 35% in sequenced plant genomes. The multiplex CRISPR system is the tool of choice for creating targeted TAG deletions. Here, we show that up to ~80% of CRISPR-mediated TAG knockout alleles in Arabidopsis and rice are deletion-inversion (delinver) bi-alleles, which are easily misidentified as homozygous deletion alleles by routine PCR-based genotyping. This can lead to misinterpretation of experimental data and production of progenies with genetic heterogeneity in an unnoticed manner. In ~2,650 transgenic events, delinver mutation frequencies are predominantly correlated with deletion frequencies but unrelated to chromosomal locations or deletion sizes. Delinver mutations also occur frequently at genomic non-TAG loci during multiplexed CRISPR editing. Our work raises the alarm about delinver mutations as common unwanted products of targeted TAG deletions in plants and helps prevent false interpretation of plant TAG functions due to this hidden genotype issue.

Type-II 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-II metacaspases (MCs) constitute the proteolytic enzymes to mediate PROPEP processing in Arabidopsis. In protoplasts, co-expression of PROPEP1 with type-II 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 Botrytis cinerea resistance are severely impaired in the mc4/5/6/7 quadruple-mutant plants. Taken together, our study identifies the type-II 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.