Advancements in the Analysis of the Arabidopsis Plasma Membrane Proteome

The plasma membrane (PM) regulates diverse processes essential to plant growth, development, and survival in an ever-changing environment. In addition to maintaining normal cellular homeostasis and plant nutrient status, PM proteins perceive and respond to a myriad of environmental cues. Here we review recent advances in the analysis of the plant PM proteome with a focus on the model plant Arabidopsis thaliana. Due to membrane heterogeneity, hydrophobicity, and low relative abundance, analysis of the PM proteome has been a special challenge. Various experimental techniques to enrich PM proteins and different protein and peptide separation strategies have facilitated the identification of thousands of integral and membrane-associated proteins. Numerous classes of proteins are present at the PM with diverse biological functions. PM microdomains have attracted much attention. However, it still remains a challenge to characterize these cell membrane compartments. Dynamic changes in the PM proteome in response to different biotic and abiotic stimuli are highlighted. Future prospects for PM proteomics research are also discussed

Three previously characterized resistances to yellow rust are encoded by a single locus Wtk1.

The wild emmer wheat (Triticum turgidum ssp. dicoccoides; WEW) yellow (stripe) rust resistance genes Yr15, YrG303, and YrH52 were discovered in natural populations from different geographic locations. They all localize to chromosome 1B but were thought to be non-allelic based on differences in resistance response. We recently cloned Yr15 as a Wheat Tandem Kinase 1 (WTK1) and show here that these three resistance loci co-segregate in fine-mapping populations and share an identical full-length genomic sequence of functional Wtk1. Independent ethyl methanesulfonate (EMS)-mutagenized susceptible yrG303 and yrH52 lines carried single nucleotide mutations in Wtk1 that disrupted function. A comparison of the mutations for yr15, yrG303, and yrH52 mutants showed that while key conserved residues were intact, other conserved regions in critical kinase subdomains were frequently affected. Thus, we concluded that Yr15-, YrG303-, and YrH52-mediated resistances to yellow rust are encoded by a single locus, Wtk1. Introgression of Wtk1 into multiple genetic backgrounds resulted in variable phenotypic responses, confirming that Wtk1-mediated resistance is part of a complex immune response network. WEW natural populations subjected to natural selection and adaptation have potential to serve as a good source for evolutionary studies of different traits and multifaceted gene networks

Regulation of reactive oxygen species during plant immunity through phosphorylation and ubiquitination of RBOHD.

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

RIN4 Functions with Plasma Membrane H+-ATPases to Regulate Stomatal Apertures during Pathogen Attack

In plants, the protein Rin4 acts with the plasma membrane H+-ATPase to regulate pathogen entry and the innate immune response, in part, through the regulation of stomatal closure

A Pathogen Secreted Protein as a Detection Marker for Citrus Huanglongbing.

The citrus industry is facing an unprecedented crisis due to Huanglongbing (HLB, aka citrus greening disease), a bacterial disease associated with the pathogen Candidatus Liberibacter asiaticus (CLas) that affects all commercial varieties. Transmitted by the Asian citrus psyllid (ACP), CLas colonizes citrus phloem, leading to reduced yield and fruit quality, and eventually tree decline and death. Since adequate curative measures are not available, a key step in HLB management is to restrict the spread of the disease by identifying infected trees and removing them in a timely manner. However, uneven distribution of CLas cells in infected trees and the long latency for disease symptom development makes sampling of trees for CLas detection challenging. Here, we report that a CLas secreted protein can be used as a biomarker for detecting HLB infected citrus. Proteins secreted from CLas cells can presumably move along the phloem, beyond the site of ACP inoculation and CLas colonized plant cells, thereby increasing the chance of detecting infected trees. We generated a polyclonal antibody that effectively binds to the secreted protein and developed serological assays that can successfully detect CLas infection. This work demonstrates that antibody-based diagnosis using a CLas secreted protein as the detection marker for infected trees offers a high-throughput and economic approach that complements the approved quantitative polymerase chain reaction-based methods to enhance HLB management programs

The Plant Pathogen Pseudomonas syringae pv. tomato Is Genetically Monomorphic and under Strong Selection to Evade Tomato Immunity

Recently, genome sequencing of many isolates of genetically monomorphic bacterial human pathogens has given new insights into pathogen microevolution and phylogeography. Here, we report a genome-based micro-evolutionary study of a bacterial plant pathogen, Pseudomonas syringae pv. tomato. Only 267 mutations were identified between five sequenced isolates in 3,543,009 nt of analyzed genome sequence, which suggests a recent evolutionary origin of this pathogen. Further analysis with genome-derived markers of 89 world-wide isolates showed that several genotypes exist in North America and in Europe indicating frequent pathogen movement between these world regions. Genome-derived markers and molecular analyses of key pathogen loci important for virulence and motility both suggest ongoing adaptation to the tomato host. A mutational hotspot was found in the type III-secreted effector gene hopM1. These mutations abolish the cell death triggering activity of the full-length protein indicating strong selection for loss of function of this effector, which was previously considered a virulence factor. Two non-synonymous mutations in the flagellin-encoding gene fliC allowed identifying a new microbe associated molecular pattern (MAMP) in a region distinct from the known MAMP flg22. Interestingly, the ancestral allele of this MAMP induces a stronger tomato immune response than the derived alleles. The ancestral allele has largely disappeared from today's Pto populations suggesting that flagellin-triggered immunity limits pathogen fitness even in highly virulent pathogens. An additional non-synonymous mutation was identified in flg22 in South American isolates. Therefore, MAMPs are more variable than expected differing even between otherwise almost identical isolates of the same pathogen strain

Pathogen Specialization

The ability to infect new hosts can drive the evolution and specialization of secreted pathogen proteins

Harnessing Effector-Triggered Immunity for Durable Disease Resistance.

Genetic control of plant diseases has traditionally included the deployment of single immune receptors with nucleotide-binding leucine-rich repeat (NLR) domain architecture. These NLRs recognize corresponding pathogen effector proteins inside plant cells, resulting in effector-triggered immunity (ETI). Although ETI triggers robust resistance, deployment of single NLRs can be rapidly overcome by pathogen populations within a single or a few growing seasons. In order to generate more durable disease resistance against devastating plant pathogens, a multitiered strategy that incorporates stacked NLRs combined with other sources of disease resistance is necessary. New genetic and genomic technologies have enabled advancements in identifying conserved pathogen effectors, isolating NLR repertoires from diverse plants, and editing plant genomes to enhance resistance. Significant advancements have also been made in understanding plant immune perception at the receptor level, which has promise for engineering new sources of resistance. Here, we discuss how to utilize recent scientific advancements in a multilayered strategy for developing more durable disease resistance

Purification and Detection of Ubiquitinated Plant Proteins Using Tandem Ubiquitin Binding Entities

The timing and amplitude of plant signaling are frequently regulated through posttranslational modification of key signaling sectors, which facilitates rapid and flexible responses. Protein ubiquitination can serve as a degradation marker, influence subcellular localization, alter protein-protein interactions, and affect protein activity. Identification of polyubiquitinated proteins has been challenging due to their rapid degradation by the proteasome or removal of modifications by deubiquitination enzymes (DUBs). Tandem ubiquitin binding entities (TUBEs) are based on ubiquitin-associated domains and protect against both proteasomal degradation and DUBs. Here, we provide a protocol for purification of ubiquitinated plant proteins using TUBEs after transient expression in Nicotiana benthamiana. This protocol can also be applied to other plants to purify multiple ubiquitinated proteins or track ubiquitination of a target protein. This methodology provides an effective method for identification of ubiquitin ligase substrates and can be coupled with TUBEs targeting specific ubiquitination linkages