Variation in microbial feature perception in the Rutaceae family with immune receptor conservation in citrus.

Although much is known about the responses of model plants to microbial features, we still lack an understanding of the extent of variation in immune perception across members of a plant family. In this work, we analyzed immune responses in Citrus and wild relatives, surveying 86 Rutaceae genotypes with differing leaf morphologies and disease resistances. We found that responses to microbial features vary both within and between members. Species in 2 subtribes, the Balsamocitrinae and Clauseninae, can recognize flagellin (flg22), cold shock protein (csp22), and chitin, including 1 feature from Candidatus Liberibacter species (csp22CLas), the bacterium associated with Huanglongbing. We investigated differences at the receptor level for the flagellin receptor FLAGELLIN SENSING 2 (FLS2) and the chitin receptor LYSIN MOTIF RECEPTOR KINASE 5 (LYK5) in citrus genotypes. We characterized 2 genetically linked FLS2 homologs from "Frost Lisbon" lemon (Citrus ×limon, responsive) and "Washington navel" orange (Citrus ×aurantium, nonresponsive). Surprisingly, FLS2 homologs from responsive and nonresponsive genotypes were expressed in Citrus and functional when transferred to a heterologous system. "Washington navel" orange weakly responded to chitin, whereas "Tango" mandarin (C. ×aurantium) exhibited a robust response. LYK5 alleles were identical or nearly identical between the 2 genotypes and complemented the Arabidopsis (Arabidopsis thaliana) lyk4/lyk5-2 mutant with respect to chitin perception. Collectively, our data indicate that differences in chitin and flg22 perception in these citrus genotypes are not the results of sequence polymorphisms at the receptor level. These findings shed light on the diversity of perception of microbial features and highlight genotypes capable of recognizing polymorphic pathogen features.

Effectors from a Bacterial Vector-Borne Pathogen Exhibit Diverse Subcellular Localization, Expression Profiles, and Manipulation of Plant Defense.

Climate change is predicted to increase the prevalence of vector-borne disease due to expansion of insect populations. 'Candidatus Liberibacter solanacearum' is a phloem-limited pathogen associated with multiple economically important diseases in solanaceous crops. Little is known about the strategies and pathogenicity factors 'Ca. L. solanacearum' uses to colonize its vector and host. We determined the 'Ca. L. solanacearum' effector repertoire by predicting proteins secreted by the general secretory pathway across four different 'Ca. L. solanacearum' haplotypes, investigated effector localization in planta, and profiled effector expression in the vector and host. The localization of 'Ca. L. solanacearum' effectors in Nicotiana spp. revealed diverse eukaryotic subcellular targets. The majority of tested effectors were unable to suppress plant immune responses, indicating they possess unique activities. Expression profiling in tomato and the psyllid Bactericera cockerelli indicated 'Ca. L. solanacearum' differentially interacts with its host and vector and can switch effector expression in response to these environments. This study reveals 'Ca. L. solanacearum' effectors possess complex expression patterns, target diverse host organelles and the majority are unable to suppress host immune responses. A mechanistic understanding of 'Ca. L. solanacearum' effector function will reveal novel targets and provide insight into phloem biology. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

XAP5 CIRCADIAN TIMEKEEPER Affects Both DNA Damage Responses and Immune Signaling in Arabidopsis.

Numerous links have been reported between immune response and DNA damage repair pathways in both plants and animals but the precise nature of the relationship between these fundamental processes is not entirely clear. Here, we report that XAP5 CIRCADIAN TIMEKEEPER (XCT), a protein highly conserved across eukaryotes, acts as a negative regulator of immunity in Arabidopsis thaliana and plays a positive role in responses to DNA damaging radiation. We find xct mutants have enhanced resistance to infection by a virulent bacterial pathogen, Pseudomonas syringae pv. tomato DC3000, and are hyper-responsive to the defense-activating hormone salicylic acid (SA) when compared to wild-type. Unlike most mutants with constitutive effector-triggered immunity (ETI), xct plants do not have increased levels of SA and retain enhanced immunity at elevated temperatures. Genetic analysis indicates XCT acts independently of NONEXPRESSOR OF PATHOGENESIS RELATED GENES1 (NPR1), which encodes a known SA receptor. Since DNA damage has been reported to potentiate immune responses, we next investigated the DNA damage response in our mutants. We found xct seedlings to be hypersensitive to UV-C and γ radiation and deficient in phosphorylation of the histone variant H2A.X, one of the earliest known responses to DNA damage. These data demonstrate that loss of XCT causes a defect in an early step of the DNA damage response pathway. Together, our data suggest that alterations in DNA damage response pathways may underlie the enhanced immunity seen in xct mutants.

Regulated Disorder: Posttranslational Modifications Control the RIN4 Plant Immune Signaling Hub.

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 posttranslational 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.

The MAP4 Kinase SIK1 Ensures Robust Extracellular ROS Burst and Antibacterial Immunity in Plants.

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.

Direct and Indirect Visualization of Bacterial Effector Delivery into Diverse Plant Cell Types during Infection.

To cause disease, diverse pathogens deliver effector proteins into host cells. Pathogen effectors can inhibit defense responses, alter host physiology, and represent important cellular probes to investigate plant biology. However, effector function and localization have primarily been investigated after overexpression in planta. Visualizing effector delivery during infection is challenging due to the plant cell wall, autofluorescence, and low effector abundance. Here, we used a GFP strand system to directly visualize bacterial effectors delivered into plant cells through the type III secretion system. GFP is a beta barrel that can be divided into 11 strands. We generated transgenic Arabidopsis thaliana plants expressing GFP1-10 (strands 1 to 10). Multiple bacterial effectors tagged with the complementary strand 11 epitope retained their biological function in Arabidopsis and tomato (Solanum lycopersicum). Infection of plants expressing GFP1-10 with bacteria delivering GFP11-tagged effectors enabled direct effector detection in planta. We investigated the temporal and spatial delivery of GFP11-tagged effectors during infection with the foliar pathogen Pseudomonas syringae and the vascular pathogen Ralstonia solanacearum Thus, the GFP strand system can be broadly used to investigate effector biology in planta.

Plant-Pathogen Effectors: Cellular Probes Interfering with Plant Defenses in Spatial and Temporal Manners.

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 Pseudomonas syringae type III effector HopD1 suppresses effector-triggered immunity, localizes to the endoplasmic reticulum, and targets the Arabidopsis transcription factor NTL9.

• Pseudomonas 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.

Phytoplasma PMU1 exists as linear chromosomal and circular extrachromosomal elements and has enhanced expression in insect vectors compared with plant hosts.

Phytoplasmas 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.

AY-WB phytoplasma secretes a protein that targets plant cell nuclei.

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.