• A detailed molecular understanding of how oomycete plant pathogens evade disease resistance is essential to inform the deployment of durable resistance (R) genes. • Map-based cloning, transient expression in planta, pathogen transformation and DNA sequence variation across diverse isolates were used to identify and characterize PiAVR2 from potato late blight pathogen Phytophthora infestans. • PiAVR2 is an RXLR-EER effector that is up-regulated during infection, accumulates at the site of haustoria formation, and is recognized inside host cells by potato protein R2. Expression of PiAVR2 in a virulent P. infestans isolate conveys a gain-of-avirulence phenotype, indicating that this is a dominant gene triggering R2-dependent disease resistance. PiAVR2 presence/absence polymorphisms and differential transcription explain virulence on R2 plants. Isolates infecting R2 plants express PiAVR2-like, which evades recognition by R2. PiAVR2 and PiAVR2-like differ in 13 amino acids, eight of which are in the C-terminal effector domain; one or more of these determines recognition by R2. Nevertheless, few polymorphisms were observed within each gene in pathogen isolates, suggesting limited selection pressure for change within PiAVR2 and PiAVR2-like. • Our results direct a search for R genes recognizing PiAVR2-like, which, deployed with R2, may exert strong selection pressure against the P. infestans population.

An emerging area in plant research focuses on antagonism between regulatory systems governing growth and immunity. Such cross talk represents a point of vulnerability for pathogens to exploit. AVR2, an RXLR effector secreted by the potato blight pathogen Phytophthora infestans, interacts with potato BSL1, a putative phosphatase implicated in growthpromoting brassinosteroid (BR) hormone signaling. Transgenic potato (Solanum tuberosum) plants expressing the effector exhibit transcriptional and phenotypic hallmarks of overactive BR signaling and show enhanced susceptibility to P. infestans. Microarray analysis was used to identify a set of BR-responsive marker genes in potato, all of which are constitutively expressed to BR-induced levels in AVR2 transgenic lines. One of these genes was a bHLH transcription factor, designated StCHL1, homologous to AtCIB1 and AtHBI1, which are known to facilitate antagonism between BR and immune responses. Transient expression of either AVR2 or CHL1 enhanced leaf colonization by P. infestans and compromised immune cell death activated by perception of the elicitin Infestin1 (INF1). Knockdown of CHL1 transcript using Virus-Induced Gene Silencing (VIGS) reduced colonization of P. infestans on Nicotiana benthamiana. Moreover, the ability of AVR2 to suppress INF1-triggered cell death was attenuated in NbCHL1-silenced plants, indicating that NbCHL1 was important for this effector activity. Thus, AVR2 exploits cross talk between BR signaling and innate immunity in Solanum species, representing a novel, indirect mode of innate immune suppression by a filamentous pathogen effector.

An understanding of the cell biology underlying the burgeoning molecular genetic and genomic knowledge of oomycete pathogenicity is essential to gain the full context of how these pathogens cause disease on plants. An intense research focus on secreted Phytophthora effector proteins, especially those containing a conserved N-terminal RXLR motif, has meant that most cell biological studies into Phytophthora diseases have focussed on the effectors and their host target proteins. While these effector studies have provided novel insights into effector secretion and host defence mechanisms, there remain many unanswered questions about fundamental processes involved in spore biology, host penetration and haustorium formation and function.

To be successful plant pathogens, microbes use "effector proteins" to manipulate host functions to their benefit. Identifying host targets of effector proteins and characterizing their role in the infection process allow us to better understand plant-pathogen interactions and the plant immune system. Yeast two-hybrid analysis and coimmunoprecipitation were used to demonstrate that the Phytophthora infestans effector AVIRULENCE 2 (PiAVR2) interacts with all three BRI1-SUPPRESSOR1-like (BSL) family members from potato (Solanum tuberosum). Transient expression of BSL1, BSL2, and BSL3 enhanced P. infestans leaf infection. BSL1 and BSL3 suppressed INFESTIN 1 elicitin-triggered cell death, showing that they negatively regulate immunity. Virusinduced gene silencing studies revealed that BSL2 and BSL3 are required for BSL1 stability and show that basal levels of immunity are increased in BSL-silenced plants. Immune suppression by BSL family members is dependent on the brassinosteroid-responsive host transcription factor CIB1/HBI1-like 1. The P. infestans effector PiAVR2 targets all three BSL family members in the crop plant S. tuberosum. These phosphatases, known for their role in growth-promoting brassinosteroid signaling, all support P. infestans virulence and thus can be regarded as susceptibility factors in late blight infection.

No abstract

S-palmitoylation (S-acylation) is emerging as an important dynamic post-translational modification of cysteine residues within proteins. Current assays for protein S-palmitoylation involve either in vivo labeling or chemical cleavage of S-palmitoyl groups to reveal a free cysteine sulfhydryl that can be subsequently labeled with an affinity handle (acyl-exchange). Assays for protein S-palmitoylation using acyl-exchange chemistry therefore require blocking of non-S-palmitoylated cysteines, typically using N-ethylmaleimide (NEM), to prevent non-specific detection. This in turn necessitates multiple precipitation-based clean-up steps to remove reagents between stages, often leading to variable sample loss, reduced signal, or protein aggregation. These combine to reduce the sensitivity, reliability, and accuracy of these assays, which also require a substantial amount of time to perform. By substituting these precipitation steps with chemical scavenging of NEM by 2,3-dimethyl-1,3-butadiene in an aqueous Diels-Alder 4+2 cyclo-addition reaction, it is possible to greatly improve sensitivity and accuracy while reducing the hands-on time and overall time required for the assay.

Receptor-like kinases (RLKs) are the largest family of proteins in plants and are responsible for perceiving the vast majority of extracellular stimuli. Thus, RLKs function in diverse processes, including sensing pathogen attacks, regulating symbiotic interactions, transducing hormone and peptide signals, and monitoring cell wall status. However, despite their fundamental role in plant biology, very few antibodies are available against RLKs, which necessitates the use of epitope tags and fluorescent protein fusions in biochemical analyses such as immunoblot analysis and intracellular visualization. Epitope tags are widely used and are typically assumed to be benign, with no influence on protein function. FLAGELLIN SENSITIVE2 (FLS2) is the receptor for bacterial flagellin and often is used as a model for RLK function. Previous work implies that carboxyl-terminal epitope fusions to FLS2 maintain protein function. Here, a detailed complementation analysis of Arabidopsis () mutant plants expressing various FLS2 C-terminal epitope fusions revealed highly variable and unpredictable FLS2-mediated signaling outputs. In addition, only one out of four FLS2 epitope fusions maintained the ability to inhibit plant growth in response to flg22 treatment comparable to that in the wild type or control untagged transgenic lines. These results raise concerns over the widespread use of RLK epitope tag fusions for functional studies. Many of the subtleties of FLS2 function, and by extension those of other RLKs, may have been overlooked or inappropriately interpreted through the use of RLK epitope tag fusions.

The post-translational lipid modifications N-myristoylation, prenylation and S-acylation are traditionally associated with increasing protein membrane affinity and localisation. However this is an over-simplification, with evidence now implicating these modifications in a variety of roles such as membrane microdomain partitioning, protein trafficking, protein complex assembly and polarity maintenance. Evidence for a regulatory role is also emerging, with changes or manipulation of lipid modifications offering a means of directly controlling various aspects of protein function. Proteomics advances have revealed an enrichment of signalling proteins in the lipid-modified proteome, potentially indicating an important role for these modifications in responding to stimuli. This review highlights some of the key themes and possible functions of lipid modification during signalling processes in plants.