Tuning the Wavelength: Manipulation of Light Signaling to Control Plant Defense.

The growth-defense trade-off in plants is a phenomenon whereby plants must balance the allocation of their resources between developmental growth and defense against attack by pests and pathogens. Consequently, there are a series of points where growth signaling can negatively regulate defenses and where defense signaling can inhibit growth. Light perception by various photoreceptors has a major role in the control of growth and thus many points where it can influence defense. Plant pathogens secrete effector proteins to manipulate defense signaling in their hosts. Evidence is emerging that some of these effectors target light signaling pathways. Several effectors from different kingdoms of life have converged on key chloroplast processes to take advantage of regulatory crosstalk. Moreover, plant pathogens also perceive and react to light in complex ways to regulate their own growth, development, and virulence. Recent work has shown that varying light wavelengths may provide a novel way of controlling or preventing disease outbreaks in plants.

Chloroplasts play a central role in facilitating MAMP-triggered immunity, pathogen suppression of immunity and crosstalk with abiotic stress.

Microbe-associated molecular pattern (MAMP)-triggered immunity (MTI) research has traditionally centred around signal transduction pathways originating from activated membrane-localized pattern recognition receptors (PRRs), culminating in nuclear transcription and posttranslational modifications. More recently, chloroplasts have emerged as key immune signalling hubs, playing a central role in integrating environmental signals. Notably, MAMP recognition induces chloroplastic reactive oxygen species (cROS) that is suppressed by pathogen effectors, which also modify the balance of chloroplast-synthesized precursors of the defence hormones, jasmonic acid, salicylic acid (SA) and abscisic acid. This study focuses on how well-characterized PRRs and coreceptors modulate chloroplast physiology, examining whether diverse signalling pathways converge to similarly modulate chloroplast function. Pretreatment of receptor mutant plants with MAMP and D(Damage)AMP peptides usually protect against effector modulation of chlorophyll fluorescence and prevent Pseudomonas syringae effector-mediated quenching of cROS and suppression of maximum dark-adapted quantum efficiency (the ratio of variable/maximum fluorescence [Fv /Fm ]). The MTI coreceptor double mutant, bak1-5/bkk1-1, exhibits a remarkable decrease in Fv /Fm compared to control plants during infection, underlining the importance of MTI-mediated signalling in chloroplast immunity. Further probing the role of the chloroplast in immunity, we unexpectedly found that even moderate changes in light intensity can uncouple plant immune signalling.

Interplay between phytohormone signalling pathways in plant defence - other than salicylic acid and jasmonic acid.

Phytohormones are essential for all aspects of plant growth, development, and immunity; however, it is the interplay between phytohormones, as they dynamically change during these processes, that is key to this regulation. Hormones have traditionally been split into two groups: growth-promoting and stress-related. Here, we will discuss and show that all hormones play a role in plant defence, regardless of current designation. We highlight recent advances in our understanding of the complex phytohormone networks with less focus on archetypal immunity-related pathways and discuss protein and transcription factor signalling hubs that mediate hormone interplay.

Evolutionarily distinct resistance proteins detect a pathogen effector through its association with different host targets.

Knowledge of the evolutionary processes which govern pathogen recognition is critical to understanding durable disease resistance. We determined how Phytophthora infestans effector PiAVR2 is recognised by evolutionarily distinct resistance proteins R2 and Rpi-mcq1. We employed yeast two-hybrid, co-immunoprecipitation, virus-induced gene silencing, transient overexpression, and phosphatase activity assays to investigate the contributions of BSL phosphatases to R2- and Rpi-mcq1-mediated hypersensitive response (R2 HR and Rpi-mcq1 HR, respectively). Silencing PiAVR2 target BSL1 compromises R2 HR. Rpi-mcq1 HR is compromised only when BSL2 and BSL3 are silenced. BSL1 overexpression increases R2 HR and compromises Rpi-mcq1. However, overexpression of BSL2 or BSL3 enhances Rpi-mcq1 and compromises R2 HR. Okadaic acid, which inhibits BSL phosphatase activity, suppresses both recognition events. Moreover, expression of a BSL1 phosphatase-dead (PD) mutant suppresses R2 HR, whereas BSL2-PD and BSL3-PD mutants suppress Rpi-mcq1 HR. R2 interacts with BSL1 in the presence of PiAVR2, but not with BSL2 and BSL3, whereas no interactions were detected between Rpi-mcq1 and BSLs. Thus, BSL1 activity and association with R2 determine recognition of PiAVR2 by R2, whereas BSL2 and BSL3 mediate Rpi-mcq1 perception of PiAVR2. R2 and Rpi-mcq1 utilise distinct mechanisms to detect PiAVR2 based on association with different BSLs, highlighting central roles of these effector targets for both disease and disease resistance.

PR1-mediated defence via C-terminal peptide release is targeted by a fungal pathogen effector.

The effector SnTox3 from Parastagonospora nodorum elicits a strong necrotic response in susceptible wheat and also interacts with wheat pathogenesis-related protein 1 (TaPR-1), although the function of this interaction in disease is unclear. Here, we dissect TaPR1 function by studying SnTox3-TaPR1 interaction and demonstrate the dual functionality of SnTox3. We utilized site-directed mutagenesis to identify an SnTox3 variant, SnTox3P173S , that was unable to interact with TaPR1 in yeast-two-hybrid assays. Additionally, using recombinant proteins we established a novel protein-mediated phenotyping assay allowing functional studies to be undertaken in wheat. Wheat leaves infiltrated with TaPR1 proteins showed significantly less disease compared to control leaves, correlating with a strong increase in defence gene expression. This activity was dependent on release of the TaCAPE1 peptide embedded within TaPR1 by an unidentified serine protease. The priming activity of TaPR1 was compromised by SnTox3 but not the noninteracting variant SnTox3P173S , and we demonstrate that SnTox3 prevents TaCAPE1 release from TaPR1 in vitro. SnTox3 independently functions to induce necrosis through recognition by Snn3 and also suppresses host defence through a direct interaction with TaPR1 proteins. Importantly, this study also advances our understanding of the role of PR1 proteins in host-microbe interactions as inducers of host defence signalling.

Chloroplast immunity illuminated.

The chloroplast has recently emerged as pivotal to co-ordinating plant defence responses and as a target of plant pathogens. Beyond its central position in oxygenic photosynthesis and primary metabolism - key targets in the complex virulence strategies of diverse pathogens - the chloroplast integrates, decodes and responds to environmental signals. The capacity of chloroplasts to synthesize phytohormones and a diverse range of secondary metabolites, combined with retrograde and reactive oxygen signalling, provides exquisite flexibility to both perceive and respond to biotic stresses. These processes also represent a plethora of opportunities for pathogens to evolve strategies to directly or indirectly target 'chloroplast immunity'. This review covers the contribution of the chloroplast to pathogen associated molecular pattern and effector triggered immunity as well as systemic acquired immunity. We address phytohormone modulation of immunity and surmise how chloroplast-derived reactive oxygen species underpin chloroplast immunity through indirect evidence inferred from genetic modification of core chloroplast components and direct pathogen targeting of the chloroplast. We assess the impact of transcriptional reprogramming of nuclear-encoded chloroplast genes during disease and defence and look at future research challenges.

Volatile Molecules Secreted by the Wheat Pathogen Parastagonospora nodorum Are Involved in Development and Phytotoxicity.

Septoria nodorum blotch is a major disease of wheat caused by the fungus Parastagonospora nodorum. Recent studies have demonstrated that secondary metabolites, including polyketides and non-ribosomal peptides, produced by the pathogen play important roles in disease and development. However, there is currently no knowledge on the composition or biological activity of the volatile organic compounds (VOCs) secreted by P. nodorum. To address this, we undertook a series of growth and phytotoxicity assays and demonstrated that P. nodorum VOCs inhibited bacterial growth, were phytotoxic and suppressed self-growth. Mass spectrometry analysis revealed that 3-methyl-1-butanol, 2-methyl-1-butanol, 2-methyl-1-propanol, and 2-phenylethanol were dominant in the VOC mixture and phenotypic assays using these short chain alcohols confirmed that they were phytotoxic. Further analysis of the VOCs also identified the presence of multiple sesquiterpenes of which four were identified via mass spectrometry and nuclear magnetic resonance as ß-elemene, α-cyperone, eudesma-4,11-diene and acora-4,9-diene. Subsequent reverse genetics studies were able to link these molecules to corresponding sesquiterpene synthases in the P. nodorum genome. However, despite extensive testing, these molecules were not involved in either of the growth inhibition or phytotoxicity phenotypes previously observed. Plant assays using mutants of the pathogen lacking the synthetic genes revealed that the identified sesquiterpenes were not required for disease formation on wheat leaves. Collectively, these data have significantly extended our knowledge of the VOCs in fungi and provided the basis for further dissecting the roles of sesquiterpenes in plant disease.

AVR2 Targets BSL Family Members, Which Act as Susceptibility Factors to Suppress Host Immunity.

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. Virus-induced 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.

Loss of AvrSr50 by somatic exchange in stem rust leads to virulence for Sr50 resistance in wheat.

Race-specific resistance genes protect the global wheat crop from stem rust disease caused by Puccinia graminis f. sp. tritici (Pgt) but are often overcome owing to evolution of new virulent races of the pathogen. To understand virulence evolution in Pgt, we identified the protein ligand (AvrSr50) recognized by the Sr50 resistance protein. A spontaneous mutant of Pgt virulent to Sr50 contained a 2.5 mega-base pair loss-of-heterozygosity event. A haustorial secreted protein from this region triggers Sr50-dependent defense responses in planta and interacts directly with the Sr50 protein. Virulence alleles of AvrSr50 have arisen through DNA insertion and sequence divergence, and our data provide molecular evidence that in addition to sexual recombination, somatic exchange can play a role in the emergence of new virulence traits in Pgt.

Emerging Insights into the Functions of Pathogenesis-Related Protein 1.

The members of the pathogenesis-related protein 1 (PR-1) family are among the most abundantly produced proteins in plants on pathogen attack, and PR-1 gene expression has long been used as a marker for salicylic acid-mediated disease resistance. However, despite considerable interest over several decades, their requirement and role in plant defence remains poorly understood. Recent reports have emerged demonstrating that PR-1 proteins possess sterol-binding activity, harbour an embedded defence signalling peptide, and are targeted by plant pathogens during host infection. These studies have re-energised the field and provided long-awaited insights into a possible PR-1 function. Here we review the current status of PR-1 proteins and discuss how these recent advances shed light on putative roles for these enigmatic proteins.

RXLR Effector AVR2 Up-Regulates a Brassinosteroid-Responsive bHLH Transcription Factor to Suppress Immunity.

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 growth-promoting 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.

Production of small cysteine-rich effector proteins in Escherichia coli for structural and functional studies.

Although the lifestyles and infection strategies of plant pathogens are diverse, a prevailing feature is the use of an arsenal of secreted proteins, known as effectors, which aid in microbial infection. In the case of eukaryotic filamentous pathogens, such as fungi and oomycetes, effector proteins are typically dissimilar, at the protein sequence level, to known protein families and functional domains. Consequently, we currently have a limited understanding of how fungal and oomycete effectors promote disease. Protein biochemistry and structural biology are two methods that can contribute greatly to the understanding of protein function. Both techniques are dependent on obtaining proteins that are pure and functional, and generally require the use of heterologous recombinant protein expression systems. Here, we present a general scheme and methodology for the production and characterization of small cysteine-rich (SCR) effectors utilizing Escherichia coli expression systems. Using this approach, we successfully produced cysteine-rich effectors derived from the biotrophic fungal pathogen Melampsora lini and the necrotrophic fungal pathogen Parastagonospora nodorum. Access to functional recombinant proteins facilitated crystallization and functional experiments. These results are discussed in the context of a general workflow that may serve as a template for others interested in understanding the function of SCR effector(s) from their plant pathogen(s) of interest.

Wheat PR-1 proteins are targeted by necrotrophic pathogen effector proteins.

Recent studies have identified that proteinaceous effectors secreted by Parastagonospora nodorum are required to cause disease on wheat. These effectors interact in a gene-for-gene manner with host-dominant susceptibilty loci, resulting in disease. However, whilst the requirement of these effectors for infection is clear, their mechanisms of action remain poorly understood. A yeast-two-hybrid library approach was used to search for wheat proteins that interacted with the necrotrophic effector SnTox3. Using this strategy we indentified an interaction between SnTox3 and the wheat pathogenicity-related protein TaPR-1-1, and confirmed it by in-planta co-immunprecipitation. PR-1 proteins represent a large family (23 in wheat) of proteins that are upregulated early in the defence response; however, their function remains ellusive. Interestingly, the P. nodorum effector SnToxA has recently been shown to interact specifically with TaPR-1-5. Our analysis of the SnTox3-TaPR-1 interaction demonstrated that SnTox3 can interact with a broader range of TaPR-1 proteins. Based on these data we utilised homology modeling to predict, and validate, regions on TaPR-1 proteins that are likely to be involved in the SnTox3 interaction. Precipitating from this work, we identified that a PR-1-derived defence signalling peptide from the C-terminus of TaPR-1-1, known as CAPE1, enhanced the infection of wheat by P. nodorum in an SnTox3-dependent manner, but played no role in ToxA-mediated disease. Collectively, our data suggest that P. nodorum has evolved unique effectors that target a common host-protein involved in host defence, albeit with different mechanisms and potentially outcomes.

Differential effector gene expression underpins epistasis in a plant fungal disease.

Fungal effector-host sensitivity gene interactions play a key role in determining the outcome of septoria nodorum blotch disease (SNB) caused by Parastagonospora nodorum on wheat. The pathosystem is complex and mediated by interaction of multiple fungal necrotrophic effector-host sensitivity gene systems. Three effector sensitivity gene systems are well characterized in this pathosystem; SnToxA-Tsn1, SnTox1-Snn1 and SnTox3-Snn3. We tested a wheat mapping population that segregated for Snn1 and Snn3 with SN15, an aggressive P. nodorum isolate that produces SnToxA, SnTox1 and SnTox3, to study the inheritance of sensitivity to SnTox1 and SnTox3 and disease susceptibility. Interval quantitative trait locus (QTL) mapping showed that the SnTox1-Snn1 interaction was paramount in SNB development on both seedlings and adult plants. No effect of the SnTox3-Snn3 interaction was observed under SN15 infection. The SnTox3-Snn3 interaction was however, detected in a strain of SN15 in which SnTox1 had been deleted (tox1-6). Gene expression analysis indicates increased SnTox3 expression in tox1-6 compared with SN15. This indicates that the failure to detect the SnTox3-Snn3 interaction in SN15 is due - at least in part - to suppressed expression of SnTox3 mediated by SnTox1. Furthermore, infection of the mapping population with a strain deleted in SnToxA, SnTox1 and SnTox3 (toxa13) unmasked a significant SNB QTL on 2DS where the SnTox2 effector sensitivity gene, Snn2, is located. This QTL was not observed in SN15 and tox1-6 infections and thus suggesting that SnToxA and/or SnTox3 were epistatic. Additional QTLs responding to SNB and effectors sensitivity were detected on 2AS1 and 3AL.

Surveying the potential of secreted antimicrobial peptides to enhance plant disease resistance.

Antimicrobial peptides (AMPs) are natural products found across diverse taxa as part of the innate immune system against pathogen attacks. Some AMPs are synthesized through the canonical gene expression machinery and are called ribosomal AMPs. Other AMPs are assembled by modular enzymes generating nonribosomal AMPs and harbor unusual structural diversity. Plants synthesize an array of AMPs, yet are still subject to many pathogen invasions. Crop breeding programs struggle to release new cultivars in which complete disease resistance is achieved, and usually such resistance becomes quickly overcome by the targeted pathogens which have a shorter generation time. AMPs could offer a solution by exploring not only plant-derived AMPs, related or unrelated to the crop of interest, but also non-plant AMPs produced by bacteria, fungi, oomycetes or animals. This review highlights some promising candidates within the plant kingdom and elsewhere, and offers some perspectives on how to identify and validate their bioactivities. Technological advances, particularly in mass spectrometry (MS) and nuclear magnetic resonance (NMR), have been instrumental in identifying and elucidating the structure of novel AMPs, especially nonribosomal peptides which cannot be identified through genomics approaches. The majority of non-plant AMPs showing potential for plant disease immunity are often tested using in vitro assays. The greatest challenge remains the functional validation of candidate AMPs in plants through transgenic experiments, particularly introducing nonribosomal AMPs into crops.

The past, present and future of secondary metabolite research in the Dothideomycetes.

The Dothideomycetes represents a large and diverse array of fungi in which prominent plant pathogens are over-represented. Species within the Cochliobolus, Alternaria, Pyrenophora and Mycosphaerella (amongst others) all cause diseases that threaten food security in many parts of the world. Significant progress has been made over the past decade in understanding how some of these pathogens cause disease at a molecular level. It is reasonable to suggest that much of this progress can be attributed to the increased availability of genome sequences. However, together with revealing mechanisms of pathogenicity, these genome sequences have also highlighted the capacity of the Dothideomycetes to produce an extensive array of secondary metabolites, far greater than originally thought. Indeed, it is now clear that we appear to have only scratched the surface to date in terms of the identification of secondary metabolites produced by these fungi. In the first half of this review, we examine the current status of secondary metabolite research in the Dothideomycetes and highlight the diversity of the molecules discovered thus far, in terms of both structure and biological activity. In the second part of this review, we survey the emerging techniques and technologies that will be required to shed light on the vast array of secondary metabolite potential that is encoded within these genomes. Experimental design, analytical chemistry and synthetic biology are all discussed in the context of how they will contribute to this field.

Host protein BSL1 associates with Phytophthora infestans RXLR effector AVR2 and the Solanum demissum Immune receptor R2 to mediate disease resistance.

Plant pathogens secrete effector proteins to modulate plant immunity and promote host colonization. Plant nucleotide binding leucine-rich repeat (NB-LRR) immunoreceptors recognize specific pathogen effectors directly or indirectly. Little is known about how NB-LRR proteins recognize effectors of filamentous plant pathogens, such as Phytophthora infestans. AVR2 belongs to a family of 13 sequence-divergent P. infestans RXLR effectors that are differentially recognized by members of the R2 NB-LRR family in Solanum demissum. We report that the putative plant phosphatase BSU-LIKE PROTEIN1 (BSL1) is required for R2-mediated perception of AVR2 and resistance to P. infestans. AVR2 associates with BSL1 and mediates the interaction of BSL1 with R2 in planta, possibly through the formation of a ternary complex. Strains of P. infestans that are virulent on R2 potatoes express an unrecognized form, Avr2-like (referred to as A2l). A2L can still interact with BSL1 but does not promote the association of BSL1 with R2. Our findings show that recognition of the P. infestans AVR2 effector by the NB-LRR protein R2 requires the putative phosphatase BSL1. This reveals that, similar to effectors of phytopathogenic bacteria, recognition of filamentous pathogen effectors can be mediated via a host protein that interacts with both the effector and the NB-LRR immunoreceptor.

Presence/absence, differential expression and sequence polymorphisms between PiAVR2 and PiAVR2-like in Phytophthora infestans determine virulence on R2 plants.

• 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.

Healthy Living Cambridge Kids: a community-based participatory effort to promote healthy weight and fitness.

The objective of this study was to assess the impact of a community-based healthy weight intervention on child weight and fitness. Cambridge Public Schools (CPS) have monitored BMI and fitness annually since 2000. Annual increases of overweight and obesity from 2000 (37.0%) to 2004 (39.1%), triggered a multidisciplinary team of researchers, educators, health care, and public health professionals to mobilize environmental and policy interventions. Guided by the social-ecological model and community-based participatory research (CBPR) principles, the team developed and implemented Healthy Living Cambridge Kids (HLCK), a multicomponent intervention targeting community, school, family, and individuals. The intervention included city policies and community awareness campaigns; physical education (PE) enhancements, food service reforms, farm-to-school-to-home programs; and family outreach and "BMI and fitness reports". Baseline (2004) to follow-up (2007) evaluation design assessed change in children's weight and fitness status. A cohort of 1,858 K-5th grade children participated: 37.3% black, 14.0% Hispanic, 37.1% white, 10.2% Asian, 1.7% other race; 43.3% were lower income. BMI z-score (0.67-0.63 P < 0.001) and proportion obese (20.2-18.0% P < 0.05) decreased, and mean number of fitness tests (0-5) passed increased (3.7-3.9 P < 0.001). Whereas black and Hispanic children were more likely to be obese at baseline (27.0 and 28.5%, respectively) compared with white (12.6%) and Asian (14.3%) children, obesity among all race/ethnicity groups declined. Concurrent with a 3-year community intervention, modest improvements in obesity and fitness were observed among CPS children from baseline to follow-up. The CBPR approach facilitated sustaining policies and program elements postintervention in this diverse community.