Closely related pathogens may differ dramatically in host range, but the molecular, genetic, and evolutionary basis for these differences remains unclear. In many Gram- negative bacteria, including the phytopathogen Pseudomonas syringae, type III effectors (TTEs) are essential for pathogenicity, instrumental in structuring host range, and exhibit wide diversity between strains. To capture the dynamic nature of virulence gene repertoires across P. syringae, we screened 11 diverse strains for novel TTE families and coupled this nearly saturating screen with the sequencing and assembly of 14 phylogenetically diverse isolates from a broad collection of diseased host plants. TTE repertoires vary dramatically in size and content across all P. syringae clades; surprisingly few TTEs are conserved and present in all strains. Those that are likely provide basal requirements for pathogenicity. We demonstrate that functional divergence within one conserved locus, hopM1, leads to dramatic differences in pathogenicity, and we demonstrate that phylogenetics-informed mutagenesis can be used to identify functionally critical residues of TTEs. The dynamism of the TTE repertoire is mirrored by diversity in pathways affecting the synthesis of secreted phytotoxins, highlighting the likely role of both types of virulence factors in determination of host range. We used these 14 draft genome sequences, plus five additional genome sequences previously reported, to identify the core genome for P. syringae and we compared this core to that of two closely related non-pathogenic pseudomonad species. These data revealed the recent acquisition of a 1 Mb megaplasmid by a sub-clade of cucumber pathogens. This megaplasmid encodes a type IV secretion system and a diverse set of unknown proteins, which dramatically increases both the genomic content of these strains and the pan-genome of the species.

Plants and animals deploy intracellular immune receptors that perceive specific pathogen effector proteins and microbial products delivered into the host cell. We demonstrate that the ADR1 family of Arabidopsis nucleotide-binding leucine-rich repeat (NB-LRR) receptors regulates accumulation of the defense hormone salicylic acid during three different types of immune response: (i) ADRs are required as "helper NB-LRRs" to transduce signals downstream of specific NB-LRR receptor activation during effector-triggered immunity; (ii) ADRs are required for basal defense against virulent pathogens; and (iii) ADRs regulate microbial-associated molecular pattern-dependent salicylic acid accumulation induced by infection with a disarmed pathogen. Remarkably, these functions do not require an intact P-loop motif for at least one ADR1 family member. Our results suggest that some NB-LRR proteins can serve additional functions beyond canonical, P-loop-dependent activation by specific virulence effectors, extending analogies between intracellular innate immune receptor function from plants and animals.nucleotide-binding domain and leucine-rich repeat-containing protein receptors | plant immune system | effector-triggered immunity | microbial-associated molecular pattern-triggered immunity P lants respond to attempted microbial infection with a twotiered immune system. In the first tier, extracellular pattern recognition receptors (PRRs) bind conserved microbial-associated molecular pattern (MAMP) ligands, activating a complex host response that results in MAMP-triggered immunity (MTI). Successful pathogens deploy suites of virulence effectors that delay or suppress MTI, allowing infection. In the second tier, plant intracellular immune receptors of the nucleotide-binding leucinerich repeat (NB-LRR) protein family can be activated either by direct binding of effectors or, alternatively, by effector action on an associated target protein that generates a "modified-self" molecule (1, 2). Effector-mediated NB-LRR activation results in effectortriggered immunity (ETI), a rapid and high-amplitude output significantly overlapping with MTI (1). ETI is typically accompanied by the hypersensitive cell death response (HR), limited to the site of pathogen attack. Both MTI and some cases of NB-LRRmediated ETI require the salicylic acid (SA)-signaling molecule as a downstream mediator of transcriptional output responses (3, 4).Plant NB-LRR proteins belong to the STAND (signal transduction ATPases with numerous domains) superfamily, which includes the animal apoptotic proteins Apaf-1/CED4 and innate immune receptors of the nucleotide-binding domain and leucinerich repeat-containing proteins (NLR) family (5). Animal NLRs are activated by MAMPs and by modified-self molecules in the form of danger-associated molecular patterns (6) and regulate inflammasome activation, autophagy, and cell death (7). STAND protein functions require an intact P-loop motif (GxxxxGKT/S) that coordinates ATP binding. STAND proteins are molecular switches that toggle fr...

Summary The Arabidopsis NB-LRR immune receptor RPM1 recognizes the Pseudomonas syringae type III effectors AvrB or AvrRpm1 to mount an immune response. Although neither effector is itself a kinase, AvrRpm1 and AvrB are known to target Arabidopsis RIN4, a negative regulator of basal plant defense, for phosphorylation. We show that RIN4 phosphorylation activates RPM1. RIN4142–176 is necessary, and with appropriate localization sequences, sufficient to support effector-triggered RPM1 activation, with the threonine residue at position 166 being critical. Phosphomimic substitutions at T166 cause effector-independent RPM1 activation. RIN4 T166 is phosphorylated in vivo in the presence of AvrB or AvrRpm1. RIN4 mutants that lose interaction with AvrB cannot be co-immunoprecipitated with RPM1. This defines a common interaction platform required for RPM1 activation by phosphorylated RIN4 in response to pathogenic effectors. Conservation of an analogous threonine across all RIN4-like proteins suggests a key function for this residue beyond the regulation of RPM1.

Detection of pathogens by plants is mediated by intracellular nucleotide-binding site leucine-rich repeat (NLR) receptor proteins. NLR proteins are defined by their stereotypical multidomain structure: an N-terminal Toll-interleukin receptor (TIR) or coiled-coil (CC) domain, a central nucleotide-binding (NB) domain, and a C-terminal leucine-rich repeat (LRR). The plant innate immune system contains a limited NLR repertoire that functions to recognize all potential pathogens. We isolated Response to the bacterial type III effector protein HopBA1 (RBA1), a gene that encodes a TIR-only protein lacking all other canonical NLR domains. RBA1 is sufficient to trigger cell death in response to HopBA1. We generated a crystal structure for HopBA1 and found that it has similarity to a class of proteins that includes esterases, the hemebinding protein ChaN, and an uncharacterized domain of Pasteurella multocida toxin. Self-association, coimmunoprecipitation with HopBA1, and function of RBA1 require two previously identified TIR-TIR dimerization interfaces. Although previously described as distinct in other TIR proteins, in RBA1 neither of these interfaces is sufficient when the other is disrupted. These data suggest that oligomerization of RBA1 is required for function. Our identification of RBA1 demonstrates that "truncated" NLRs can function as pathogen sensors, expanding our understanding of both receptor architecture and the mechanism of activation in the plant immune system. plant immunity | NLR | Toll-interleukin-1 receptor homology domain | oligomerization | type III secretion

The nucleotide-binding domain leucine-rich repeat proteins (NLRs) represent the major class of intracellular innate immune receptors in plants and animals. Understanding their functions is a major challenge in immunology. This review highlights recent efforts toward elucidating NLR functions in human and plants. We compare unconventional aspects of NLR proteins across the two kingdoms. We review recent advances describing P-loop independent activation, nuclear-cytoplasmic trafficking, oligomerization and multimerization requirements for signaling, and for expanded functions beyond pathogen recognition by several NLR proteins.