Kangbiao Qi

Lysin Motif–Containing Proteins LYP4 and LYP6 Play Dual Roles in Peptidoglycan and Chitin Perception in Rice Innate Immunity

This work characterizes the roles of two LysM-containing proteins, LYP4 and LYP6, in rice innate immunity. It finds that they act as unique dual-function pattern recognition receptors that can recognize microbe-associated molecular patterns from bacteria and fungi. Plant innate immunity relies on successful detection of microbe-associated molecular patterns (MAMPs) of invading microbes via pattern recognition receptors (PRRs) at the plant cell surface. Here, we report two homologous rice (Oryza sativa) lysin motif–containing proteins, LYP4 and LYP6, as dual functional PRRs sensing bacterial peptidoglycan (PGN) and fungal chitin. Live cell imaging and microsomal fractionation consistently revealed the plasma membrane localization of these proteins in rice cells. Transcription of these two genes could be induced rapidly upon exposure to bacterial pathogens or diverse MAMPs. Both proteins selectively bound PGN and chitin but not lipopolysaccharide (LPS) in vitro. Accordingly, silencing of either LYP specifically impaired PGN- or chitin- but not LPS-induced defense responses in rice, including reactive oxygen species generation, defense gene activation, and callose deposition, leading to compromised resistance against bacterial pathogen Xanthomonas oryzae and fungal pathogen Magnaporthe oryzae. Interestingly, pretreatment with excess PGN dramatically attenuated the alkalinization response of rice cells to chitin but not to flagellin; vice versa, pretreatment with chitin attenuated the response to PGN, suggesting that PGN and chitin engage overlapping perception components in rice. Collectively, our data support the notion that LYP4 and LYP6 are promiscuous PRRs for PGN and chitin in rice innate immunity.

MpAsr encodes an intrinsically unstructured protein and enhances osmotic tolerance in transgenic Arabidopsis.

Abscisic acid-, stress- and ripening (ASR) -induced proteins are plant-specific proteins whose expression is up-regulated under abiotic stresses or during fruit ripening. In this study, we characterized an ASR protein from plantain to explore its physiological roles under osmotic stress. The expression pattern of MpAsr gene shows that MpAsr gene changed little at the mRNA level, while the MpASR protein accumulates under osmotic treatment. Through bioinformatic-based predictions, circular dichroism spectrometry, and proteolysis and heat-stability assays, we determined that the MpASR protein is an intrinsically unstructured protein in solution. We demonstrated that the hydrophilic MpASR protein could protect l-lactate dehydrogenase (l-LDH) from cold-induced aggregation. Furthermore, heterologous expression of MpAsr in Escherichia coli and Arabidopsis enhanced the tolerance of transformants to osmotic stress. Transgenic 35S::MpAsr Arabidopsis seeds had a higher germination frequency than wild-type seeds under unfavorable conditions. At the physiological level, 35S::MpAsr Arabidopsis showed increased soluble sugars and decreased cell membrane damage under osmotic stress. Thus, our results suggest that the MpASR protein may act as an osmoprotectant and water-retaining molecule to help cell adjustment to water deficit caused by osmotic stress.

HYPERSENSITIVE TO HIGH LIGHT1 Interacts with LOW QUANTUM YIELD OF PHOTOSYSTEM II1 and Functions in Protection of Photosystem II from Photodamage in Arabidopsis

This work identified the thylakoid protein HYPERSENSITIVE TO HIGH LIGHT1 (HHL1), specifically conserved in land plants, as an important regulator of photosynthesis and photoprotection. Together with LOW QUANTUM YIELD OF PHOTOSYSTEM II1, HHL1 regulates the repair and reassembly of photosystem II complex under high-light conditions in Arabidopsis. Under high-irradiance conditions, plants must efficiently protect photosystem II (PSII) from damage. In this study, we demonstrate that the chloroplast protein HYPERSENSITIVE TO HIGH LIGHT1 (HHL1) is expressed in response to high light and functions in protecting PSII against photodamage. Arabidopsis thaliana hhl1 mutants show hypersensitivity to high light, drastically decreased PSII photosynthetic activity, higher nonphotochemical quenching activity, a faster xanthophyll cycle, and increased accumulation of reactive oxygen species following high-light exposure. Moreover, HHL1 deficiency accelerated the degradation of PSII core subunits under high light, decreasing the accumulation of PSII core subunits and PSII–light-harvesting complex II supercomplex. HHL1 primarily localizes in the stroma-exposed thylakoid membranes and associates with the PSII core monomer complex through direct interaction with PSII core proteins CP43 and CP47. Interestingly, HHL1 also directly interacts, in vivo and in vitro, with LOW QUANTUM YIELD OF PHOTOSYSTEM II1 (LQY1), which functions in the repair and reassembly of PSII. Furthermore, the hhl1 lqy1 double mutants show increased photosensitivity compared with single mutants. Taken together, these results suggest that HHL1 forms a complex with LQY1 and participates in photodamage repair of PSII under high light.

Comparative characterization, expression pattern and function analysis of the 12-oxo-phytodienoic acid reductase gene family in rice

The 12-oxo-phytodienoic acid reductases (OPRs) belong to the old yellow enzyme family of flavoenzymes and form multiple subfamilies in angiosperm plants. In our previous study, a comparative genomic analysis showed that five OPR subfamilies (subs. I–V) occur in monocots, and two subfamilies (subs. I and II) in dicots. Here, a comparative study of five OsOPR genes, representing five subfamilies (I–V) in rice, was performed to provide insights into OPR biochemical properties and physiological importance. Comparative analysis of the three-dimensional structure by homology modeling indicated all five OsOPR proteins contained a highly conserved backbone with (α/β)8-barrels, while two middle variable regions (MVR i and ii) were also detected and defined. Analysis of enzymatic characteristics revealed that all five OsOPR fusion proteins exhibit distinct substrate specificity. Different catalytic activity was observed using racemic OPDA and trans-2-hexen-1-al as substrates, suggesting OsOPR family genes participate in two main branches of the octadecanoid pathway, including the allene oxide synthase and hydroperoxide lyase pathways which regulate various developmental processes and/or defense responses. The transcript profiles of five OsOPR genes exhibited strong tissue-specific and inducible expression patterns under abiotic stress, hormones and plant wounding treatments. Furthermore, the transcriptions of OsOPR04-1 (OsOPR11) and OsOPR08-1 (OsOPR7), representing subs. I and II, respectively, were observed in all six selected tissues and with all above-stress treatments. This suggests that these two subfamilies play an important role during different developmental stages and in response to stresses; while the expressions of OsOPR06-1 (OsOPR6), OsOPR01-1 (OsOPR10) and OsOPR02-1 (OsOPR8), representing subs. III, IV and V respectively, were strongly up-regulated with abscisic acid (ABA) and indoleacetic acid (IAA) treatments in roots, suggesting these three subfamilies play an important role in responding to hormones especially ABA and IAA signals in roots.

Musa paradisica RCI complements AtRCI and confers Na+ tolerance and K+ sensitivity in Arabidopsis.

The mechanisms involved in Na⁺/K⁺ uptake and extrusion are important in plant salt tolerance. In this study, we investigated the physiological role of a plasma membrane (PM)-localized protein, MpRCI, from plantain in transgenic Arabidopsis under NaCl and KCl stress and determined its effect on PM fluidity and H⁺-ATPase activity. The MpRCI gene exhibited high homology to the AtRCI2 gene family in Arabidopsis and was therefore able to complement for loss of the yeast AtRCI2-related PMP3 gene. Results of phenotypic espial and atomic emission spectrophotometer (AES) assays indicated that MpRCI overexpression in the AtRCI2A knockout mutant with reduced shoot Na⁺ and increased K⁺ exhibited increased Na⁺-tolerance and K⁺-sensitivity under NaCl or KCl treatments, respectively. Furthermore, comparisons of PM fluidity and H⁺-ATPase activity in shoots, with expression or absence of MpRCI/AtRCI2A expression under NaCl or KCl stress, showed MpRCI maintained PM fluidity and H⁺-ATPase activity under stress conditions. Results suggest that MpRCI plays an essential role in Na⁺/K⁺ flux in plant cells.

Cu2+ triggers reversible aggregation of a disordered His-rich dehydrin MpDhn12 from Musa paradisiaca.

Copper is an essential nutrient, but it is toxic in excess. Here, we cloned and characterized a His-rich low molecular weight dehydrin from Musa paradisiaca, MpDhn12. Analysis by circular dichroism (CD) spectra and a thermal stability assay showed that MpDhn12 is an intrinsically disordered protein, and immobilized-metal affinity chromatography (IMAC) analysis revealed that MpDhn12 can bind Cu(2+) both in vitro and in vivo. Interestingly, MpDhn12 aggregated under excess Cu(2+) conditions, and the aggregation was reversible and impaired by histidine modification with diethylpyrocarbonate (DEPC), while the disordered structure of another dehydrin ERD14 (as a control) was not changed. Furthermore, MpDhn12 could complement the copper-sensitive phenotype of yeast mutant Δsod1. These results together suggested that MpDhn12 may take part in buffering copper levels through chelation and formation of aggregates in excess Cu(2+) conditions. To the best of our knowledge, it is the first report that a dehydrin interchanged between disordered and aggregated state triggered by copper.

OsLYP4 and OsLYP6 play critical roles in rice defense signal transduction.

Plant innate immunity relies on successful detection of trespassing pathogens through recognizing their microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) at the cell surface. We recently reported two rice lysin motif (LysM)-containing proteins, OsLYP4 and OsLYP6, as dual functional PRRs sensing bacterial peptidoglycan (PGN) and fungal chitin. Here we further demonstrated the important roles of OsLYP4 and OsLYP6 in rice defense signaling, as silencing of either LYP impaired the defense marker gene activation induced by either bacterial pathogen Xanthomonas oryzaecola or fungal pathogen Magnaporthe oryzae. Moreover, we found that OsLYP4 and OsLYP6 could form homo- and hetero-dimers, and could interact with CEBiP, suggesting an unexpected complexity of chitin perception in rice.