Summary During infection, plant pathogens secrete effector proteins to facilitate colonization. In comparison with our knowledge of bacterial effectors, the current understanding of how fungal effectors function is limited. In this study, we show that the effector AvrL567‐A from the flax rust fungus Melampsora lini interacts with a flax cytosolic cytokinin oxidase, LuCKX1.1, using both yeast two‐hybrid and in planta bimolecular fluorescence assays. Purified LuCKX1.1 protein shows catalytic activity against both N6‐(Δ2‐isopentenyl)‐adenine (2iP) and trans ‐zeatin (tZ) substrates. Incubation of LuCKX1.1 with AvrL567‐A results in increased catalytic activity against both substrates. The crystal structure of LuCKX1.1 and docking studies with AvrL567‐A indicate that the AvrL567 binding site involves a flexible surface‐exposed region that surrounds the cytokinin substrate access site, which may explain its effect in modulating LuCKX1.1 activity. Expression of AvrL567‐A in transgenic flax plants gave rise to an epinastic leaf phenotype consistent with hormonal effects, although no difference in overall cytokinin levels was observed. We propose that, during infection, plant pathogens may differentially modify the levels of extracellular and intracellular cytokinins.
Since the first report of the plasmid-mediated, colistin-resistant gene, mcr-1, nine mcr genes and their subvariants have been identified. The spreading scope of mcr-1~10 varies greatly, suggesting that mcr-1~10 may have different evolutionary advantages. Depending on MCR family phylogeny, mcr-6 is highly similar to mcr-1 and -2, and mcr-7~10 are highly similar to mcr-3 and -4. We compared the expression effects of MCR-1~5 on bacteria of common physiological background. The MCR-1-expressing strain showed better growth than did MCR-2~5-expressing strains in the presence of colistin. LIVE/DEAD staining analysis revealed that MCR-3~5 expression exerted more severe fitness burdens on bacteria than did MCR-1 and -2. Bacteria expressing MCRs except MCR-2 showed enhanced virulence with increased epithelial penetration ability determined by trans-well model (p < 0.05). Enhanced virulence was also observed in the Galleria mellonella model, which may have resulted from bacterial membrane damage and different levels of lipopolysaccharide (LPS) release due to MCR expression. Collectively, MCR-1-expressing strain showed the best survival advantage of MCR-1~5-expressing strains, which may partly explain the worldwide distribution of mcr-1. Our results suggested that MCR expression may cause increased bacterial virulence, which is alarming, and further attention will be needed to focus on the control of infectious diseases caused by mcr-carrying pathogens.
Plant intracellular immune receptors known as NLR (Nucleotide-binding Leucine-rich repeat, NB-LRR) proteins confer resistance and cause cell death upon recognition of cognate effector proteins from pathogens. Plant NLRs contain a variable N-terminal domain: a Toll/interleukin-1 receptor (TIR) domain or a coiled-coil (CC) domain or an RPW8 (Resistance to Powdery Mildew 8)-like CC (CCR) domain. TIR-NLR, CC-NLR and CCR-NLR are known as TNL, CNL and RNL, respectively. TNLs and CNLs recognize pathogen effectors to activate cell death and defense responses, thus are regarded as sensor NLRs. RNLs are required downstream of TNLs to activate cell death and defense responses, thus are regarded as helper NLRs. Previous studies show that some TNLs form tetrameric resistosome as NAD+ cleaving enzymes to transduce signal, while some CNLs form pentameric resistosome with undefined biochemical function. Two recent breakthrough studies show that activated CNL and RNL function as Ca2+ channel to cause cell death and defense responses and provide a completely new insight into the downstream signaling events of CNL and TNL pathways.
ABSTRACT Insects secret chemosensory proteins (CSPs) into plant cells as potential effector proteins during feeding. The molecular mechanisms underlying how CSPs activate plant immunity remain largely unknown. We show that CSPs from six distinct insect orders induce dwarfism when overexpressed in Nicotiana benthamiana . Agrobacterium‐mediated transient expression of Nilaparvata lugens CSP11 (NlCSP11) triggered cell death and plant dwarfism, both of which were dependent on ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), N requirement gene 1 (NRG1) and SENESCENCE‐ASSOCIATED GENE 101 (SAG101), indicating the activation of effector‐triggered immunity (ETI) in N. benthamiana . Overexpression of NlCSP11 led to stronger systemic resistance against Pseudomonas syringae DC3000 lacking effector HopQ1‐1 and tobacco mosaic virus, and induced higher accumulation of salicylic acid (SA) in uninfiltrated leaves compared to another effector XopQ that is recognized by a Toll‐interleukin‐1 receptor (TIR) domain nucleotide‐binding leucine‐rich repeat receptor (TNL) called ROQ1 in N. benthamiana . Consistently, NlCSP11‐induced dwarfism and systemic resistance, but not cell death, were abolished in N. benthamiana transgenic line expressing the SA‐degrading enzyme NahG . Through large‐scale virus‐induced gene silencing screening, we identified a TNL protein that mediates the recognition of CSPs (RCSP), including aphid effector MP10 that triggers resistance against aphids in N. benthamiana . Co‐immunoprecipitation, bimolecular fluorescence complementation and AlphaFold2 prediction unveiled an interaction between NlCSP11 and RCSP. Interestingly, RCSP does not contain the conserved catalytic glutamic acid in the TIR domain, which is required for TNL function. Our findings point to enhanced ETI and systemic resistance by a TNL protein via hyperactivation of the SA pathway. Moreover, RCSP is the first TNL identified to recognize an insect effector.
Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) analyzed to date oligomerize and form resistosomes upon activation to initiate immune responses. Some NLRs are encoded in tightly linked co-regulated head-to-head genes whose products function together as pairs. We uncover the oligomerization requirements for different Arabidopsis paired CHS3-CSA1 alleles. These pairs form resting-state heterodimers that oligomerize into complexes distinct from NLRs analyzed previously. Oligomerization requires both conserved and allele-specific features of the respective CHS3 and CSA1 Toll-like interleukin-1 receptor (TIR) domains. The receptor kinases BAK1 and BIRs inhibit CHS3-CSA1 pair oligomerization to maintain the CHS3-CSA1 heterodimer in an inactive state. Our study reveals that paired NLRs hetero-oligomerize and likely form a distinctive “dimer of heterodimers” and that structural heterogeneity is expected even among alleles of closely related paired NLRs.
This study investigated the effects of xylo-oligosaccharide (XOS) and flavomycin (FLA) on the performance and immune function of broiler chickens.A total of 150 ArborAcres broilers were randomly divided into three groups and fed for 6 weeks from 1 day of age in cascade cages.The diets of each test group were 1) a basal diet, 2) the basal diet supplemented with 2 mg/kg FLA, and 3) the basal diet supplemented with 2 mg/kg XOS.At 21 and 42 days, the growth performance index values and short-chain fatty acid (SCFA) concentrations in the cecum were quantified.Furthermore, immunoglobulin G (IgG) and plasma interleukin 2 (IL-2) as well as mRNA expression of LPS-Induced TNF-alpha Factor (LITAF), Toll-like receptor-5 (TLR5) and interferon gamma (IFNγ) in the jejunum were quantified.The results showed that administration of XOS or FLA to chickens significantly improved the average daily gain.Supplementation with XOS increased acetate and butyrate in the cecum, while FLA supplementation increased propionate in the cecum.An increase in plasma IgG was observed in XOS-fed 21-day-old broilers, but FLA supplementation decreased IgG in the plasma of 42-day-old broilers and increased plasma IL-2.Furthermore, FLA or XOS supplementation downregulated mRNA expression of IFNγ, LITAF and TLR5.The above data suggest that addition of XOS and FLA to the diet could improve the growth performance of broilers, reduce the expression of cytokine genes by stimulating SCFA.
Significance Pathogen recognition first occurs at the plasma membrane, where receptor-like kinases perceive pathogen-derived molecules and initiate immune responses. To abrogate this immune response, pathogens evolved effector proteins that act as virulence factors, often following delivery to the host cell. Plants evolved intracellular receptors, known as NOD-like receptors (NLRs), to detect effectors, thereby ensuring activation of effector-triggered immunity. However, despite their importance in immunity, the molecular mechanisms underlying effector recognition and subsequent immune activation by membrane-localized NLRs remain to be fully elucidated. Our analyses reveal the importance of and need for self-association and the coordinated interplay of specific domains and conserved residues for NLR activity. This could provide strategies for crop improvement, contributing to effective, environmentally friendly, and sustainable solutions for future agriculture.
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