AKSF1 Isolated From the Rice-Virulent Strain Acidovorax avenae K1 Is a Novel Effector That Suppresses PAMP-Triggered Immunity in Rice.

Microbial pathogens deliver effectors into plant cells to suppress plant immune responses and modulate host metabolism in order to support infection processes. We sought to determine if the Acidovorax avenae rice-virulent K1 strain can suppress pathogen-associated molecular pattern-triggered immunity (PTI) induced by flagellin isolated from the rice-avirulent N1141 strain. The flagellin-triggered PTI, including H2O2 generation, callose deposition, and expression of several immune-related genes were strongly suppressed in K1 preinoculated cultured rice cells in a type III secretion system (T3SS)-dependent manner. By screening 4,562 transposon-tagged mutants based on their suppression ability, we found that 156 transposon-tagged K1 mutants lost the ability to suppress PTI induction. Mutant sequence analysis, comprehensive expression analysis using RNA sequencing, and the prediction of secretion through T3SS showed that a protein named A. avenae K1 suppression factor 1 (AKSF1) suppresses flagellin-triggered PTI in rice. Translocation of AKSF1 protein into rice cells is dependent on the T3SS during infection, an AKSF1-disruption mutant lost the ability to suppress PTI responses, and expression of AKSF1 in the AKSF1-disruption mutant complemented the suppression activity. When AKSF1-disruption mutants were inoculated into the host rice plant, reduction of the disease symptoms and suppression of bacterial growth were observed. Taken together, our results demonstrate that AKSF1 is a novel effector that can suppress the PTI in a host rice plant.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law. 2021.

CD2-1, the C-Terminal Region of Flagellin, Modulates the Induction of Immune Responses in Rice.

Flagellin from the rice avirulent N1141 strain of Acidovorax avenae functions as a pathogen-associated molecular pattern (PAMP) and induces PAMP-triggered immunity (PTI) in rice. To study the recognition mechanism of flagellin in rice, we attempted to define one or more regions of the flagellin protein required to activate the PTI response. Based on domain classification, we produced four fragments of N1141 flagellin: N-terminal D0, D1. and D2 domains (ND0-2), N-terminal D2, D3, and C-terminal D2 domains (ND2-CD2), C-terminal D2, D1, and D0 domains (CD2-0), and C-terminal D2 and D1 domains (CD2-1). The C-terminal CD2-1 and CD2-0 fragments induced PTI responses in cultured rice cells. Synthetic flg22, which is sufficient to produce the flagellin response in Arabidopsis, and the N-terminal flagellin fragments containing flg22 region elicited very weak immune responses in rice. OsFLS2, the rice ortholog of AtFLS2, which mediates flg22 recognition, was not involved in CD2-0 or CD2-1 recognition in rice. In addition, CD2-0 triggered resistance to coinfection with pathogenic bacteria. Taken together, these data suggest that rice mainly recognizes flagellin CD2-1 by a receptor distinct from OsFLS2 and that this epitope recognition leads to PTI responses.

Two distinct EF-Tu epitopes induce immune responses in rice and Arabidopsis.

Plants sense potential pathogens by recognizing conserved pathogen-associated molecular patterns (PAMPs) that cause PAMP-triggered immunity (PTI). We previously reported that rice recognizes flagellin from the rice-incompatible N1141 strain of Acidovorax avenae and subsequently induces immune responses. Cell extracts isolated from flagellin-deficient N1141 (Δfla1141) still induced PTI responses, suggesting that Δfla1141 possesses an additional PAMP distinct from flagellin. Here, we show that elongation factor Tu (EF-Tu), one of the most abundant bacterial proteins, acts as a PAMP in rice and causes several PTI responses. In Brassicaceae species, EF-Tu and an N-acetylated peptide comprising the first 18 amino acids of the N-terminus, termed elf18, are fully active as inducers of PTI responses. By contrast, elf18 did not cause any immune responses in rice, whereas an EF-Tu middle region comprising Lys176 to Gly225, termed EFa50, is fully active as a PAMP in rice. In the leaves of rice plants, EF-Tu induced H2O2 generation and callose deposition, and also triggered resistance to coinfection with pathogenic bacteria. Taken together, these data demonstrate that rice recognizes EFa50, which is distinct from elf18, and that this epitope induces PTI responses.

Frameshift Mutation Confers Function as Virulence Factor to Leucine-Rich Repeat Protein from Acidovorax avenae.

Many plant pathogens inject type III (T3SS) effectors into host cells to suppress host immunity and promote successful infection. The bacterial pathogen Acidovorax avenae causes brown stripe symptom in many species of monocotyledonous plants; however, individual strains of each pathogen infect only one host species. T3SS-deleted mutants of A. avenae K1 (virulent to rice) or N1141 (virulent to finger millet) caused no symptom in each host plant, suggesting that T3SS effectors are involved in the symptom formation. To identify T3SS effectors as virulence factors, we performed whole-genome and predictive analyses. Although the nucleotide sequence of the novel leucine-rich repeat protein (Lrp) gene of N1141 had high sequence identity with K1 Lrp, the amino acid sequences of the encoded proteins were quite different due to a 1-bp insertion within the K1 Lrp gene. An Lrp-deleted K1 strain (KΔLrp) did not cause brown stripe symptom in rice (host plant for K1); by contrast, the analogous mutation in N1141 (NΔLrp) did not interfere with infection of finger millet. In addition, NΔLrp retained the ability to induce effector-triggered immunity (ETI), including hypersensitive response cell death and expression of ETI-related genes. These data indicated that K1 Lrp functions as a virulence factor in rice, whereas N1141 Lrp does not play a similar role in finger millet. Yeast two-hybrid screening revealed that K1 Lrp interacts with oryzain α, a pathogenesis-related protein of the cysteine protease family, whereas N1141 Lrp, which contains LRR domains, does not. This specific interaction between K1 Lrp and oryzain α was confirmed by Bimolecular fluorescence complementation assay in rice cells. Thus, K1 Lrp protein may have acquired its function as virulence factor in rice due to a frameshift mutation.

Glycan moiety of flagellin in Acidovorax avenae K1 prevents the recognition by rice that causes the induction of immune responses.

Abstract Recognition of pathogen-associated molecular patterns (PAMPs) such as flagellin, a main component of the bacterial flagellum, constitutes the first layer of plant immunity and is referred to as PAMP-triggered immunity (PTI). The rice avirulent N1141 strain of gram-negative phytopathogenic bacterium, Acidovorax avenae, induces PTI including H2O2 generation, while flagellin from the rice virulent K1 strain of A. avenae does not induce these immune responses. Mass spectrometry analyses revealed that total 1,600-Da and 2,150-Da of glycan residues were present on the flagellins from N1141 and K1, respectively. A deglycosylated K1 flagellin induced immune responses in the same manner as N1141 flagellin, suggesting that the glycan in K1 flagellin prevent epitope recognition in rice. We identified three genes in K1 flagella operon, which regulate structural modification of glycan in K1 flagellin. The immature glycan-attached flagellin from three genes deletion mutant, KΔ3FG, induced H2O2 generation in cultured rice cells, whereas the K1 mature-type flagellin did not cause a detectable increase in H2O2. The data indicate that the immature glycan of flagellin from KΔ3FG cannot prevent the epitope recognition in rice.