Hormones orchestrate the physiology of organisms. Measuring the activity of defense hormone-responsive genes can help understanding immune signaling and facilitate breeding for plant health. However, ...different from model species like Arabidopsis, genes that respond to defense hormones salicylic acid (SA) and jasmonic acid (JA) have not been disclosed in the soybean crop. We performed global transcriptome analyses to fill this knowledge gap. Upon exogenous application, endogenous levels of SA and JA increased in leaves. SA predominantly activated genes linked to systemic acquired resistance and defense signaling whereas JA mainly activated wound response-associated genes. In general, SA-responsive genes were activated earlier than those responding to JA. Consistent with the paradigm of biotrophic pathogens predominantly activating SA responses, free SA and here identified most robust SA marker genes GmNIMIN1, GmNIMIN1.2 and GmWRK40 were induced upon inoculation with Phakopsora pachyrhizi, whereas JA marker genes did not respond to infection with the biotrophic fungus. Spodoptera exigua larvae caused a strong accumulation of JA-Ile and JA-specific mRNA transcripts of GmBPI1, GmKTI1 and GmAAT whereas neither free SA nor SA-marker gene transcripts accumulated upon insect feeding. Our study provides molecular tools for monitoring the dynamic accumulation of SA and JA, e.g. in a given stress condition.
Cytoskeleton remodelling is a crucial process in determining the polarity of dividing and growing plant cells, as well as during interactions with the environment. Nothing is currently known about ...the proteins, which regulate actin remodelling during interactions with invading pathogens. The biotrophic powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh) invades susceptible barley (Hordeum vulgare L.) by penetrating epidermal cells, which remain intact during fungal development. In contrast, resistant host plants prevent infection by inhibiting penetration through apoplastic mechanisms, which require focusing defence reactions on the site of attack. We stained actin filaments in a susceptible Mlo-genotype and a near-isogenic race-non-specifically resistant barley mlo5-mutant genotype using fluorescence-labelled phalloidin after chemical fixation. This revealed that the actin cytoskeleton is differentially reorganized in susceptible and resistant hosts challenged by Bgh. Actin filaments were polarized towards the sites of attempted penetration in the resistant host, whereas when susceptible hosts were penetrated, a more subtle reorganization took place around fungal haustoria. Strong actin filament focusing towards sites of fungal attack was closely associated with successful prevention of penetration. Actin focusing was less frequent and seemingly delayed in susceptible wild-type barley expressing the susceptibility factor MLO. Additionally, single cell overexpression of a constitutively activated RAC/ROP G-protein, CA RACB, another potential host susceptibility factor and hypothetical actin cytoskeleton regulator, partly inhibited actin reorganization when under attack from Bgh, whereas knockdown of RACB promoted actin focusing. We conclude that RACB and, potentially, MLO are host proteins involved in the modulation of actin reorganization and cell polarity in the interaction of barley with Bgh.
Summary
Small monomeric G‐proteins of the plant ras (rat sarcome oncogene product) related C3 botulinum toxin substrate (RAC)/Rho of plants (ROP) family are molecular switches in signal transduction ...of many cellular processes. RAC/ROPs regulate hormone effects, subcellular gradients of Ca2+, the organisation of the actin cytoskeleton and the production of reactive oxygen intermediates. Therefore, we followed a genetic bottom‐up strategy to study the role of these proteins during the interaction of barley (Hordeum vulgare L.) with the fungal biotrophic pathogen Blumeria graminis f.sp. hordei (Bgh). We identified six barley RAC/ROP proteins and studied their gene expression. Five out of six Rac/Rop genes were expressed constitutively in the leaf epidermis, which is the site of interaction with Bgh. None of the genes showed enhancement of mRNA abundance after inoculation with Bgh. After microprojectile mediated transformation of single barley epidermal cells with constitutively activated mutant RAC/ROP proteins, we found an RAC/ROP‐specific enhancement of pathogen accessibility, tagging HvRACB, HvRAC3 and HvROP6 as host proteins potentially involved in the establishment of susceptibility to Bgh. Confocal laser scanning microscopy (CLSM) of green fluorescent protein (GFP):HvRAC/ROP‐transformed cells revealed varying strengths of plasma membrane association of barley RAC/ROPs. The C‐terminal CAAX motif for presumable prenylation or the C‐terminal hypervariable region (HVR), respectively, were required for membrane association of the RAC/ROPs. Proper intracellular localisation was essential for HvRACB and HvRAC3 function. Together, our data support the view that different paths of host signal transduction via RAC/ROP G‐proteins are involved in processes supporting parasitic entry into epidermal host cells.
Small GTP-binding proteins such as those from the RAC family are cytosolic signal transduction proteins that often are involved in processing of extracellular stimuli. Plant RAC proteins are ...implicated in regulation of plant cell architecture, secondary wall formation, meristem signaling, and defense against pathogens. We isolated a RacB homolog from barley (Hordeum vulgare) to study its role in resistance to the barley powdery mildew fungus (Blumeria graminis f.sp. hordei). RacB was constitutively expressed in the barley epidermis and its expression level was not strongly influenced by inoculation with B. graminis. However, after biolistic bombardment of barley leaf segments with RacB-double-stranded RNA, sequence-specific RNA interference with RacB function inhibited fungal haustorium establishment in a cell-autonomous and genotype-specific manner. Mutants compromised in function of the Mlo wild-type gene and the Ror1 gene (genotype mlo5 ror1) that are moderately susceptible to B. graminis showed no alteration in powdery mildew resistance upon RacB-specific RNA interference. Thus, the phenotype, induced by RacB-specific RNA interference, was apparently dependent on the same processes as mlo5-mediated broad resistance, which is suppressed by ror1. We conclude that an RAC small GTP-binding protein is required for successful fungal haustorium establishment and that this function may be linked to MLO-associated functions.
BAX inhibitor-1 (BI-1) proteins have been characterized as suppressors of programmed cell death in mammals and plants. The barley BI-1 is a suppressor of nonspecific background resistance and ...mlo-mediated penetration resistance to the biotrophic fungal pathogen Blumeria graminis f. sp. hordei when overexpressed in epidermal cells of barley. We report here that BI-1 expression is also slightly up-regulated during interaction with the inappropriate wheat pathogen Blumeria graminis f. sp. tritici. Significantly, over-expression of BI-1 in single epidermal cells of barley by microprojectile-mediated transformation rendered cells susceptible to penetration by inappropriate B. graminis f. sp. tritici. The degree of transgene-induced accessibility to B. graminis f. sp. tritici was thereby similar to the effect achieved by overexpression of the defense suppressor gene Mlo and could not be further enhanced by double expression of both BI-1 and Mlo. Confocal laser scanning microscopy was used to locate a functional green fluorescing GFP:BI-1 fusion protein in endomembranes and the nuclear envelope of barley epidermal cells. Together, enhanced expression of barley BI-1 suppresses penetration resistance to B. graminis f. sp. tritici, linking barley nonhost resistance with cell death regulation.
Small RAC/ROP-family G proteins regulate development and stress responses in plants. Transient overexpression and RNA interference experiments suggested that the barley (Hordeum vulgare) RAC/ROP ...protein RACB is involved in susceptibility to the powdery mildew fungus Blumeria graminis f. sp. hordei. We created transgenic barley plants expressing the constitutively activated RACB mutant racb-G15V under control of the maize (Zea mays) ubiquitin 1 promoter. Individuals of the T1 generation expressing racb-G15V were significantly more susceptible to B. graminis when compared to segregating individuals that did not express racb-G15V. Additionally, racb-G15V-expressing plants showed delayed shoot development from the third leaf stage on, downward rolled leaves, and stunted roots. Expression of racb-G15V decreased photosynthetic CO2-assimilation rates and transpiration of nonstressed leaves. In contrast, racb-G15V-expressing barley leaves, when detached from water supply, showed increased water loss and enhanced transpiration. Water loss was associated with reduced responsiveness to abscisic acid in regard to transpiration when compared to segregants not expressing racb-G15V. Hence, RACB might be a common signaling element in response to both biotic and abiotic stress.
The highly conserved zinc-finger protein, CTCF, is a candidate tumor suppressor protein that binds to highly divergent DNA sequences. CTCF has been connected to multiple functions in chromatin ...organization and gene regulation including chromatin insulator activity and transcriptional enhancement and silencing. Here we show that CTCF harbors several autonomous repression domains. One of these domains, the zinc-finger cluster, silences transcription in all cell types tested and binds directly to the co-repressor SIN3A. Two distinct regions of SIN3A, the PAH3 domain and the extreme C-terminal region, bind independently to this zinc-finger cluster. Analysis of nuclear extract from HeLa cells revealed that CTCF is also capable of retaining functional histone deacetylase activity. Furthermore, the ability of regions of CTCF to retain deacetylase activity correlates with the ability to bind to SIN3A and to repress gene activity. We suggest that CTCF driven repression is mediated in part by the recruitment of histone deacetylase activity by SIN3A.
Fungal pathogens pose a major challenge to global crop production. Crop varieties that resist disease present the best defence and offer an alternative to chemical fungicides. Exploiting durable ...nonhost resistance (NHR) for crop protection often requires identification and transfer of NHR‐linked genes to the target crop. Here, we identify genes associated with NHR of Arabidopsis thaliana to Phakopsora pachyrhizi, the causative agent of the devastating fungal disease called Asian soybean rust. We transfer selected Arabidopsis NHR‐linked genes to the soybean host and discover enhanced resistance to rust disease in some transgenic soybean lines in the greenhouse. Interspecies NHR gene transfer thus presents a promising strategy for genetically engineered control of crop diseases.
Summary
The fungus Phakopsora pachyrhizi (Pp) causes Asian soybean rust (SBR) disease which provokes tremendous losses in global soybean production. Pp is mainly controlled with synthetic fungicides ...to which the fungus swiftly develops fungicide resistance. To substitute or complement synthetic fungicides in Asian soybean rust control, we aimed to identify antifungal metabolites in Arabidopsis which is not a host for Pp. Comparative transcriptional and metabolic profiling of the Pp‐inoculated Arabidopsis non‐host and the soybean host revealed induction of phenylpropanoid metabolism‐associated genes in both species but activation of scopoletin biosynthesis only in the resistant non‐host. Scopoletin is a coumarin and an antioxidant. In vitro experiments disclosed fungistatic activity of scopoletin against Pp, associated with reduced accumulation of reactive oxygen species (ROS) in fungal pre‐infection structures. Non‐antioxidant and antioxidant molecules including coumarins with a similar structure to scopoletin were inactive or much less effective at inhibiting fungal accumulation of ROS and germination of Pp spores. When sprayed onto Arabidopsis leaves, scopoletin also suppressed the formation of Pp pre‐infection structures and penetration of the plant. However, scopoletin neither directly activated defence nor did it prime Arabidopsis for enhanced defence, therefore emphasizing fungistatic activity as the exclusive mode of action of scopoletin against Pp. Because scopletin also protected soybean from Pp infection, the coumarin may serve as a natural fungicide or as a lead for the development of near‐to‐nature fungicides against Asian soybean rust.
Significance Statement
Plants with non‐host resistance to a given pathogen harbour promising traits for effective plant protection. We report that upon inoculation with the Asian soybean rust‐causing fungus Phakopsora pachyrhizi the coumarin scopoletin specifically accumulates in the Arabidopsis non‐host, but not the soybean host. Because exogenously applied scopoletin scavenges ROS in the pre‐infection structures of P. pachyrhizi, inhibits spore germination, and protects soybean from Asian soybean rust the coumarin may qualify as a natural fungicide.
Little is known about the function of host factors involved in disease susceptibility. The barley (Hordeum vulgare) ROP (RHO of plants) G-protein RACB is required for full susceptibility of the leaf ...epidermis to invasion by the biotrophic fungus Blumeria graminis f. sp hordei. Stable transgenic knockdown of RACB reduced the ability of barley to accommodate haustoria of B. graminis in intact epidermal leaf cells and to form hairs on the root epidermis, suggesting that RACB is a common element of root hair outgrowth and ingrowth of haustoria in leaf epidermal cells. We further identified a barley MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN (MAGAP1) interacting with RACB in yeast and in planta. Fluorescent MAGAP1 decorated cortical microtubules and was recruited by activated RACB to the cell periphery. Under fungal attack, MAGAP1-labeled microtubules built a polarized network at sites of successful defense. By contrast, microtubules loosened where the fungus succeeded in penetration. Genetic evidence suggests a function of MAGAP1 in limiting susceptibility to penetration by B. graminis. Additionally, MAGAP1 influenced the polar organization of cortical microtubules. These results add to our understanding of how intact plant cells accommodate fungal infection structures and suggest that RACB and MAGAP1 might be antagonistic players in cytoskeleton organization for fungal entry.
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