Plants possess large arsenals of immune receptors capable of recognizing all pathogen classes. To cause disease, pathogenic organisms must be able to overcome physical barriers, suppress or evade immune perception, and derive nutrients from host tissues. Consequently, to facilitate some of these processes, pathogens secrete effector proteins that promote colonization. This review covers recent advances in the field of effector biology, focusing on conserved cellular processes targeted by effectors from diverse pathogens. The ability of effectors to facilitate pathogen entry into the host interior, suppress plant immune perception, and alter host physiology for pathogen benefit is discussed. Pathogens also deploy effectors in a spatial and temporal manner, depending on infection stage. Recent advances have also enhanced our understanding of effectors acting in specific plant organs and tissues. Effectors are excellent cellular probes that facilitate insight into biological processes as well as key points of vulnerability in plant immune signaling networks.

The fully sequenced genome of aster yellows phytoplasma strain witches' broom (AY-WB; Candidatus Phytoplasma asteris) was mined for the presence of genes encoding secreted proteins based on the presence of N-terminal signal peptides (SP). We identified 56 secreted AY-WB proteins (SAP). These SAP are candidate effector proteins potentially involved in interaction with plant and insect cell components. One of these SAP, SAP11, contains an N-terminal SP sequence and a eukaryotic bipartite nuclear localization signal (NLS). Transcripts for SAP11 were detected in AY-WB-infected plants. Yellow fluorescence protein (YFP)-tagged SAP11 accumulated in Nicotiana benthamiana cell nuclei, whereas the nuclear targeting of YFP-tagged SAP11 mutants with disrupted NLS was inhibited. The nuclear transport of YFP-SAP11 was also inhibited in N. benthamiana plants in which the expression of importin alpha was knocked down using virus-induced gene silencing (VIGS). Furthermore, SAP11 was detected by immunocytology in nuclei of young sink tissues of China aster plants infected with AY-WB. In summary, this work shows that AY-WB phytoplasma produces a protein that targets the nuclei of plant host cells; this protein is a potential phytoplasma effector that may alter plant cell physiology.

Production of reactive oxygen species (ROS) is critical for successful activation of immune responses against pathogen infection. The plant NADPH oxidase RBOHD is a primary player in ROS production during innate immunity. However, how RBOHD is negatively regulated remains elusive. Here we show that RBOHD is regulated by C-terminal phosphorylation and ubiquitination. Genetic and biochemical analyses reveal that the PBL13 receptor-like cytoplasmic kinase phosphorylates RBOHD's C-terminus and two phosphorylated residues (S862 and T912) affect RBOHD activity and stability, respectively. Using protein array technology, we identified an E3 ubiquitin ligase PIRE (PBL13 interacting RING domain E3 ligase) that interacts with both PBL13 and RBOHD. Mimicking phosphorylation of RBOHD (T912D) results in enhanced ubiquitination and decreased protein abundance. PIRE and PBL13 mutants display higher RBOHD protein accumulation, increased ROS production, and are more resistant to bacterial infection. Thus, our study reveals an intricate post-translational network that negatively regulates the abundance of a conserved NADPH oxidase.

Microbial patterns are recognized by cell-surface receptors to initiate pattern-triggered immunity (PTI) in plants. Receptor-like cytoplasmic kinases (RLCKs), such as BIK1, and calcium-dependent protein kinases (CPKs) are engaged during PTI to activate the NADPH oxidase RBOHD for reactive oxygen species (ROS) production. It is unknown whether protein kinases besides CPKs and RLCKs participate in RBOHD regulation. We screened mutants in all ten Arabidopsis MAP4 kinases (MAP4Ks) and identified the conserved MAP4K SIK1 as a positive regulator of PTI. sik1 mutants were compromised in their ability to elicit the ROS burst in response to microbial features and exhibited compromised PTI to bacterial infection. SIK1 directly interacts with, phosphorylates, and stabilizes BIK1 in a kinase activity-dependent manner. Furthermore, SIK1 directly interacts with and phosphorylates RBOHD upon flagellin perception. Thus, SIK1 positively regulates immunity by stabilizing BIK1 and activating RBOHD to promote the extracellular ROS burst.

SummaryPseudomonas syringae type III effectors are known to suppress plant immunity to promote bacterial virulence. However, the activities and targets of these effectors are not well understood.We used genetic, molecular, and cell biology methods to characterize the activities, localization, and target of the HopD1 type III effector in Arabidopsis.HopD1 contributes to P. syringae virulence in Arabidopsis and reduces effector-triggered immunity (ETI) responses but not pathogen-associated molecular pattern-triggered immunity (PTI) responses. Plants expressing HopD1 supported increased growth of ETI-inducing P. syringae strains compared with wild-type Arabidopsis. We show that HopD1 interacts with the membrane-tethered Arabidopsis transcription factor NTL9 and demonstrate that this interaction occurs at the endoplasmic reticulum (ER). A P. syringae hopD1 mutant and ETI-inducing P. syringae strains exhibited enhanced growth on Arabidopsis ntl9 mutant plants. Conversely, growth of P. syringae strains was reduced in plants expressing a constitutively active NTL9 derivative, indicating that NTL9 is a positive regulator of plant immunity. Furthermore, HopD1 inhibited the induction of NTL9-regulated genes during ETI but not PTI.HopD1 contributes to P. syringae virulence in part by targeting NTL9, resulting in the suppression of ETI responses but not PTI responses and the promotion of plant pathogenicity.

SummaryPhytoplasmas replicate intracellularly in plants and insects and are dependent on both hosts for dissemination in nature. Phytoplasmas have small genomes lacking genes for major metabolic pathways. Nevertheless, their genomes harbour multicopy gene clusters that were named potential mobile units (PMUs). PMU1 is the largest most complete repeat among the PMUs in the genome of Aster Yellows phytoplasma strain Witches' Broom (AY-WB). PMU1 is c. 20 kb in size and contains 21 genes encoding DNA replication and predicted membrane-targeted proteins. Here we show that AY-WB has a chromosomal linear PMU1 (L-PMU1) and an extrachromosomal circular PMU1 (C-PMU1). The C-PMU1 copy number was consistently higher by in average approximately fivefold in insects compared with plants and PMU1 gene expression levels were also considerably higher in insects indicating that C-PMU1 synthesis and expression are regulated. We found that the majority of AY-WB virulence genes lie on chromosomal PMU regions that have similar gene content and organization as PMU1 providing evidence that PMUs contribute to phytoplasma host adaptation and have integrated into the AY-WB chromosome.

RIN4 is an intensively studied immune regulator in Arabidopsis and is involved in perception of microbial features outside and bacterial effectors inside plant cells. Furthermore, RIN4 is conserved in land plants and is targeted for posttranslational modifications by several virulence proteins from the bacterial pathogen Pseudomonas syringae. Despite the important roles of RIN4 in plant immune responses, its molecular function is not known. RIN4 is an intrinsically disordered protein (IDP), except at regions where pathogen-induced posttranslational modifications take place. IDP act as hubs for protein complex formation due to their ability to bind to multiple client proteins and, thus, are important players in signal transduction pathways. RIN4 is known to associate with multiple proteins involved in immunity, likely acting as an immune-signaling hub for the formation of distinct protein complexes. Genetically, RIN4 is a negative regulator of immunity, but diverse post-translational modifications can either enhance its negative regulatory function or, on the contrary, render it a potent immune activator. In this review, we describe the structural domains of RIN4 proteins, their intrinsically disordered regions, posttranslational modifications, and highlight the implications that these features have on RIN4 function. In addition, we will discuss the potential role of plasma membrane subdomains in mediating RIN4 protein complex formations.

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