Powdery mildew effectors AVRA1 and BEC1016 target the ER J-domain protein HvERdj3B required for immunity in barley.

The barley powdery mildew fungus, Blumeria hordei (Bh), secretes hundreds of candidate secreted effector proteins (CSEPs) to facilitate pathogen infection and colonization. One of these, CSEP0008, is directly recognized by the barley nucleotide-binding leucine-rich-repeat (NLR) receptor MLA1 and therefore is designated AVRA1. Here, we show that AVRA1 and the sequence-unrelated Bh effector BEC1016 (CSEP0491) suppress immunity in barley. We used yeast two-hybrid next-generation interaction screens (Y2H-NGIS), followed by binary Y2H and in planta protein-protein interactions studies, and identified a common barley target of AVRA1 and BEC1016, the endoplasmic reticulum (ER)-localized J-domain protein HvERdj3B. Silencing of this ER quality control (ERQC) protein increased Bh penetration. HvERdj3B is ER luminal, and we showed using split GFP that AVRA1 and BEC1016 translocate into the ER signal peptide-independently. Overexpression of the two effectors impeded trafficking of a vacuolar marker through the ER; silencing of HvERdj3B also exhibited this same cellular phenotype, coinciding with the effectors targeting this ERQC component. Together, these results suggest that the barley innate immunity, preventing Bh entry into epidermal cells, requires ERQC. Here, the J-domain protein HvERdj3B appears to be essential and can be regulated by AVRA1 and BEC1016. Plant disease resistance often occurs upon direct or indirect recognition of pathogen effectors by host NLR receptors. Previous work has shown that AVRA1 is directly recognized in the cytosol by the immune receptor MLA1. We speculate that the AVRA1 J-domain target being inside the ER, where it is inapproachable by NLRs, has forced the plant to evolve this challenging direct recognition.

Barley endosomal MONENSIN SENSITIVITY1 is a target of the powdery mildew effector CSEP0162 and plays a role in plant immunity.

Encasements formed around haustoria and biotrophic hyphae as well as hypersensitive reaction (HR) cell death are essential plant immune responses to filamentous pathogens. In this study we examine the components that may contribute to the absence of these responses in susceptible barley attacked by the powdery mildew fungus. We find that the effector CSEP0162 from this pathogen targets plant MONENSIN SENSITIVITY1 (MON1), which is important for the fusion of multivesicular bodies to their target membranes. Overexpression of CSEP0162 and silencing of barley MON1 both inhibit encasement formation. We find that the Arabidopsis ecotype No-0 has resistance to powdery mildew, and that this is partially dependent on MON1. Surprisingly, we find the MON1-dependent resistance in No-0 not only includes an encasement response, but also an effective HR. Similarly, silencing of MON1 in barley also blocks Mla3-mediated HR-based powdery mildew resistance. Our results indicate that MON1 is a vital plant immunity component, and we speculate that the barley powdery mildew fungus introduces the effector CSEP0162 to target MON1 and hence reduce encasement formation and HR.

The evolution of plant proton pump regulation via the R domain may have facilitated plant terrestrialization.

Plasma membrane (PM) H+-ATPases are the electrogenic proton pumps that export H+ from plant and fungal cells to acidify the surroundings and generate a membrane potential. Plant PM H+-ATPases are equipped with a C­terminal autoinhibitory regulatory (R) domain of about 100 amino acid residues, which could not be identified in the PM H+-ATPases of green algae but appeared fully developed in immediate streptophyte algal predecessors of land plants. To explore the physiological significance of this domain, we created in vivo C-terminal truncations of autoinhibited PM H+­ATPase2 (AHA2), one of the two major isoforms in the land plant Arabidopsis thaliana. As more residues were deleted, the mutant plants became progressively more efficient in proton extrusion, concomitant with increased expansion growth and nutrient uptake. However, as the hyperactivated AHA2 also contributed to stomatal pore opening, which provides an exit pathway for water and an entrance pathway for pests, the mutant plants were more susceptible to biotic and abiotic stresses, pathogen invasion and water loss, respectively. Taken together, our results demonstrate that pump regulation through the R domain is crucial for land plant fitness and by controlling growth and nutrient uptake might have been necessary already for the successful water-to-land transition of plants.

The Bacillus cereus Strain EC9 Primes the Plant Immune System for Superior Biocontrol of Fusarium oxysporum.

Antibiosis is a key feature widely exploited to develop biofungicides based on the ability of biological control agents (BCAs) to produce fungitoxic compounds. A less recognised attribute of plant-associated beneficial microorganisms is their ability to stimulate the plant immune system, which may provide long-term, systemic self-protection against different types of pathogens. By using conventional antifungal in vitro screening coupled with in planta assays, we found antifungal and non-antifungal Bacillus strains that protected the ornamental plant Kalanchoe against the soil-borne pathogen Fusarium oxysporum in experimental and commercial production settings. Further examination of one antifungal and one non-antifungal strain indicated that high protection efficacy in planta did not correlate with antifungal activity in vitro. Whole-genome sequencing showed that the non-antifungal strain EC9 lacked the biosynthetic gene clusters associated with typical antimicrobial compounds. Instead, this bacterium triggers the expression of marker genes for the jasmonic and salicylic acid defence pathways, but only after pathogen challenge, indicating that this strain may protect Kalanchoe plants by priming immunity. We suggest that the stimulation of the plant immune system is a promising mode of action of BCAs for the development of novel biological crop protection products.

Bacillus cereus EC9 protects tomato against Fusarium wilt through JA/ET-activated immunity.

The mechanisms of action and the limitations of effectiveness of natural biocontrol agents should be determined in order to convert them into end products that can be used in practice. Rhizosphere Bacillus spp. protect plants from various pathogens by displaying several modes of action. However, the ability of Bacillus spp. to control plant diseases depends on the interaction between the bacteria, host, and pathogen, and the environmental conditions. We found that soil drenching of tomato plants with the non-antifungal Bacillus cereus strain EC9 (EC9) enhances plant defense against Fusarium oxysporum f. sp. lycopersici (Fol). To study the involvement of plant defense-related phytohormones in the regulation of EC9-activated protection against Fol, we conducted plant bioassays in tomato genotypes impaired in salicylic acid (SA) accumulation, jasmonic acid (JA) biosynthesis, and ethylene (ET) production, and analyzed the transcript levels of pathways-related marker genes. Our results indicate that JA/ET-dependent signaling is required for EC9-mediated protection against Fol in tomato. We provide evidence that EC9 primes tomato plants for enhanced expression of proteinase inhibitor I (PI-I) and ethylene receptor4 (ETR4). Moreover, we demonstrated that EC9 induces callose deposition in tomato roots. Understanding the involvement of defense-related phytohormones in EC9-mediated defense against Fusarium wilt has increased our knowledge of interactions between non-antifungal plant defense-inducing rhizobacteria and plants.

A holistic view on plant effector-triggered immunity presented as an iceberg model.

The immune system of plants is highly complex. It involves pattern-triggered immunity (PTI), which is signaled and manifested through branched multi-step pathways. To counteract this, pathogen effectors target and inhibit individual PTI steps. This in turn can cause specific plant cytosolic nucleotide-binding leucine-rich repeat (NLR) receptors to activate effector-triggered immunity (ETI). Plants and pathogens have many genes encoding NLRs and effectors, respectively. Yet, only a few segregate genetically as resistance (R) genes and avirulence (Avr) effector genes in wild-type populations. In an attempt to explain this contradiction, a model is proposed where far most of the NLRs, the effectors and the effector targets keep one another in a silent state. In this so-called "iceberg model", a few NLR-effector combinations are genetically visible above the surface, while the vast majority is hidden below. Besides, addressing the existence of many NLRs and effectors, the model also helps to explain why individual downregulation of many effectors causes reduced virulence and why many lesion-mimic mutants are found. Finally, the iceberg model accommodates genuine plant susceptibility factors as potential effector targets.

Barley isochorismate synthase mutant is phylloquinone-deficient, but has normal basal salicylic acid level.

Salicylic acid (SA) is an important signaling hormone in plant immunity. It can be synthesized by either the phenylpropanoid pathway or the isochorismate pathway, but mutant studies of this have been scarce in other species than Arabidopsis. Here we identified a mutation that introduced a stop-codon early in the barley gene for isochorismate synthase (ICS). We found that homozygous ics plants wilted if not sprayed with 1,4-dihydroxy-2-naphthoic acid, a precursor of phylloquinone, also synthesized via the isochorismate pathway. Interestingly, ics had unchanged SA, suggesting that the basal level of SA is synthesized via the phenylpropanoid pathway. Previous studies have failed seeing increased SA levels in barley after attack by the powdery mildew fungus, Blumeria graminis f.sp. hordei (Bgh), and indeed, we saw no changes in the interaction of ics with this fungus. Overall, we hope this mutant will be useful for other studies of SA in barley.

The isoelectric point of proteins influences their translocation to the extrahaustorial matrix of the barley powdery mildew fungus.

Many biotrophic fungal plant pathogens develop feeding structures, haustoria, inside living plant cells, which are essential for their success. Extrahaustorial membranes (EHMs) surround haustoria and delimit the extrahaustorial matrices (EHMxs). Little is known about transport mechanisms across EHMs and what properties proteins and nutrients need in order to cross these membranes. To investigate this further, we expressed fluorescent proteins in the cytosol of infected barley leaf epidermal cells after particle bombardment and investigated properties that influenced their localisation in the powdery mildew EHMx. We showed that this translocation is favoured by a neutral isoelectric point (pI) between 6.0 and 8.4. However, for proteins larger than 50 kDa, pI alone does not explain their localisation, hinting towards a more complex interplay between pI, size, and sequence properties. We discuss the possibility that an EHM translocon is involved in protein uptake into the EHMx.

Prevalence of Soil-borne Diseases in Kalanchoe blossfeldiana Reveals a Complex of Pathogenic and Opportunistic Fungi.

Greenhouse cultivation of ornamentals is subjected to a high incidence of soil-borne fungal pathogens. In Kalanchoe, these pathogens are responsible for root and stem rot, and for infection of the vascular tissue. Well-known soil-borne pathogens are believed to cause these diseases. Yet, a systematized survey of these pathogens is lacking for Kalanchoe produced commercially. Here, we studied the occurrence of soil-borne fungal pathogens associated with cultivation of Kalanchoe in Denmark and production of cuttings and stock plants in greenhouse facilities located in Turkey and Vietnam. Molecular identification of pathogens complemented mycological identification and pathogenicity testing of the soil-borne fungal pathogens. This study revealed that the fungi Corynespora cassiicola, Thielaviopsis basicola, Fusarium solani, and F. oxysporum are the most prevalent pathogens associated with root and stem rotting and wilt of Kalanchoe under the conditions studied. Furthermore, the study showed that some of the pathogens are part of an infection complex comprising both primary and opportunistic fungal species. The fact that some of the pathogens were present in some regions, while absent in others, suggests how they move between greenhouse facilities on infected plant material. This study generated important information about the soil-borne fungal complex affecting Kalanchoe, which is useful for a better understanding of the biology of the disease and for designing control strategies.

The AMSH3 ESCRT-III-Associated Deubiquitinase Is Essential for Plant Immunity.

Plant "nucleotide-binding leucine-rich repeat" receptor proteins (NLRs) detect alterations in host targets of pathogen effectors and trigger immune responses. The Arabidopsis thaliana mutant pen1 syp122 displays autoimmunity, and a mutant screen identified the deubiquitinase "associated molecule with the SH3 domain of STAM3" (AMSH3) to be required for this phenotype. AMSH3 has previously been implicated in ESCRT-mediated vacuolar targeting. Pathology experiments show that AMSH3 activity is required for immunity mediated by the CC-NLRs, RPS2 and RPM1. Co-expressing the autoactive RPM1D505V and the catalytically inactive ESCRT-III protein SKD1E232Q in Nicotiana benthamiana supports the requirement of ESCRT-associated functions for this CC-NLR-activated immunity. Meanwhile, loss of ESCRT function in A. thaliana is lethal, and we find that AMSH3 knockout-triggered seedling lethality is "enhanced disease susceptibility 1" (EDS1) dependent. Future studies may reveal whether AMSH3 is monitored by a TIR-NLR immunity receptor.

Loss of VPS9b enhances vps9a-2 phenotypes.

Plant innate immunity enables plants to defend themselves against infectious pathogens. While membrane trafficking and release of exosomes are considered vital for correct execution of innate immunity, the mechanisms behind remain elusive. Recently, we have shown that VPS9a, the general guanine-nucleotide exchange factor activating Rab5 GTPases, is required for both pre- and post-invasive immunity against powdery mildew fungi in Arabidopsis thaliana. Yet, the Arabidopsis genome contains a close homologue of VPS9a, which potentially plays specific roles in innate immunity. Here we show that this gene, VPS9b, while weakly expressed, contributes to regulating development and disease resistance, which is predominantly regulated by VPS9a. Based on these observations, we suggest that VPS9b has no specialized functionality, but rather is becoming a non-expressed pseudogene.

The plant membrane surrounding powdery mildew haustoria shares properties with the endoplasmic reticulum membrane.

Many filamentous plant pathogens place specialized feeding structures, called haustoria, inside living host cells. As haustoria grow, they are believed to manipulate plant cells to generate a specialized, still enigmatic extrahaustorial membrane (EHM) around them. Here, we focused on revealing properties of the EHM. With the help of membrane-specific dyes and transient expression of membrane-associated proteins fused to fluorescent tags, we studied the nature of the EHM generated by barley leaf epidermal cells around powdery mildew haustoria. Observations suggesting that endoplasmic reticulum (ER) membrane-specific dyes labelled the EHM led us to find that Sar1 and RabD2a GTPases bind this membrane. These proteins are usually associated with the ER and the ER/cis-Golgi membrane, respectively. In contrast, transmembrane and luminal ER and Golgi markers failed to label the EHM, suggesting that it is not a continuum of the ER. Furthermore, GDP-locked Sar1 and a nucleotide-free RabD2a, which block ER to Golgi exit, did not hamper haustorium formation. These results indicated that the EHM shares features with the plant ER membrane, but that the EHM membrane is not dependent on conventional secretion. This raises the prospect that an unconventional secretory pathway from the ER may provide this membrane's material. Understanding these processes will assist future approaches to providing resistance by preventing EHM generation.

VPS9a Activates the Rab5 GTPase ARA7 to Confer Distinct Pre- and Postinvasive Plant Innate Immunity.

Plant innate immunity can effectively prevent the proliferation of filamentous pathogens. Papilla formation at the site of attack is essential for preinvasive immunity; in postinvasive immunity, the encasement of pathogen structures inside host cells can hamper disease. Whereas papillae are highly dependent on transcytosis of premade material, little is known about encasement formation. Here, we show that endosome-associated VPS9a, the conserved guanine-nucleotide exchange factor activating Rab5 GTPases, is required for both pre- and postinvasive immunity against a nonadapted powdery mildew fungus (Blumeria graminis f. sp hordei) in Arabidopsis thaliana Surprisingly, VPS9a acts in addition to two previously well-described innate immunity components and thus represents an additional step in the regulation of how plants resist pathogens. We found VPS9a to be important for delivering membrane material to the encasement and VPS9a also plays a predominant role in postinvasive immunity. GTP-bound Rab5 GTPases accumulate in the encasement, but not the papillae, suggesting that two independent pathways form these defense structures. VPS9a also mediates defense to an adapted powdery mildew fungus, thus regulating a durable type of defense that works in both host and nonhost resistance. We propose that VPS9a plays a conserved role in organizing cellular endomembrane trafficking, required for delivery of defense components in response to powdery mildew fungi.

A Split-GFP Gateway Cloning System for Topology Analyses of Membrane Proteins in Plants.

To understand the function of membrane proteins, it is imperative to know their topology. For such studies, a split green fluorescent protein (GFP) method is useful. GFP is barrel-shaped, consisting of 11 ß-sheets. When the first ten ß-sheets (GFP1-10) and the 11th ß-sheet (GFP11) are expressed from separate genes they will self-assembly and reconstitute a fluorescent GFP protein. However, this will only occur when the two domains co-localize in the same cellular compartment. We have developed an easy-to-use Gateway vector set for determining on which side of the membrane the N- and C-termini are located. Two vectors were designed for making N- and C-terminal fusions between the membrane proteins-of-interest and GFP11, while another three plasmids were designed to express GFP1-10 in either the cytosol, the endoplasmic reticulum (ER) lumen or the apoplast. We tested functionality of the system by applying the vector set for the transmembrane domain, CNXTM, of the ER membrane protein, calnexin, after transient expression in Nicotiana benthamiana leaves. We observed GFP signal from the ER when we reciprocally co-expressed GFP11-CNXTM with GFP1-10-HDEL and CNXTM-GFP with cytosolic GFP1-10. The opposite combinations did not result in GFP signal emission. This test using the calnexin ER-membrane domain demonstrated its C-terminus to be in the cytosol and its N-terminus in the ER lumen. This result confirmed the known topology of calnexin, and we therefore consider this split-GFP system highly useful for ER membrane topology studies. Furthermore, the vector set provided is useful for detecting the topology of proteins on other membranes in the cell, which we confirmed for a plasma membrane syntaxin. The set of five Ti-plasmids are easily and efficiently used for Gateway cloning and transient transformation of N. benthamiana leaves.

The stripe rust fungal effector PEC6 suppresses pattern-triggered immunity in a host species-independent manner and interacts with adenosine kinases.

We identified a wheat stripe rust (Puccinia striiformis) effector candidate (PEC6) with pattern-triggered immunity (PTI) suppression function and its corresponding host target. PEC6 compromised PTI host species-independently. In Nicotiana benthamiana, it hampers reactive oxygen species (ROS) accumulation and callose deposition induced by Pseudomonas fluorescens. In Arabidopsis, plants expressing PEC6 were more susceptible to Pseudomonas syringae pv. tomato (Pto) DC3000 ΔAvrPto/ΔAvrPtoB. In wheat, PEC6-suppression of P. fluorescens-elicited PTI was revealed by the fact that it allowed activation of effector-triggered immunity by Pto DC3000. Knocking down of PEC6 expression by virus-mediated host-induced gene silencing decreased the number of rust pustules, uncovering PEC6 as an important pathogenicity factor. PEC6, overexpressed in plant cells without its signal peptide, was localized to the nucleus and cytoplasm. A yeast two-hybrid assay showed that PEC6 interacts with both wheat and Arabidopsis adenosine kinases (ADKs). Knocking down wheat ADK expression by virus-induced gene silencing reduced leaf growth and enhanced the number of rust pustules, indicating that ADK is important in plant development and defence. ADK plays essential roles in regulating metabolism, cytokinin interconversion and methyl transfer reactions, and our data propose a model where PEC6 may affect one of these processes by targeting ADK to favour fungal growth.

The Barley Powdery Mildew Effector Candidates CSEP0081 and CSEP0254 Promote Fungal Infection Success.

Effectors play significant roles in the success of pathogens. Recent advances in genome sequencing have revealed arrays of effectors and effector candidates from a wide range of plant pathogens. Yet, the vast majority of them remain uncharacterized. Among the ~500 Candidate Secreted Effector Proteins (CSEPs) predicted from the barley powdery mildew fungal genome, only a few have been studied and shown to have a function in virulence. Here, we provide evidence that CSEP0081 and CSEP0254 contribute to infection by the fungus. This was studied using Host-Induced Gene Silencing (HIGS), where independent silencing of the transcripts for these CSEPs significantly reduced the fungal penetration and haustoria formation rate. Both CSEPs are likely required during and after the formation of haustoria, in which their transcripts were found to be differentially expressed, rather than in epiphytic tissue. When expressed in barley leaf epidermal cells, both CSEPs appears to move freely between the cytosol and the nucleus, suggesting that their host targets locate in these cellular compartments. Collectively, our data suggest that, in addition to the previously reported effectors, the barley powdery mildew fungus utilizes these two CSEPs as virulence factors to enhance infection.

The barley powdery mildew candidate secreted effector protein CSEP0105 inhibits the chaperone activity of a small heat shock protein.

Pathogens secrete effector proteins to establish a successful interaction with their host. Here, we describe two barley (Hordeum vulgare) powdery mildew candidate secreted effector proteins, CSEP0105 and CSEP0162, which contribute to pathogen success and appear to be required during or after haustorial formation. Silencing of either CSEP using host-induced gene silencing significantly reduced the fungal haustorial formation rate. Interestingly, both CSEPs interact with the barley small heat shock proteins, Hsp16.9 and Hsp17.5, in a yeast two-hybrid assay. Small heat shock proteins are known to stabilize several intracellular proteins, including defense-related signaling components, through their chaperone activity. CSEP0105 and CSEP0162 localized to the cytosol and the nucleus of barley epidermal cells, whereas Hsp16.9 and Hsp17.5 are cytosolic. Intriguingly, only those specific CSEPs changed localization and became restricted to the cytosol when coexpressed with Hsp16.9 and Hsp17.5, confirming the CSEP-small heat shock protein interaction. As predicted, Hsp16.9 showed chaperone activity, as it could prevent the aggregation of Escherichia coli proteins during thermal stress. Remarkably, CSEP0105 compromised this activity. These data suggest that CSEP0105 promotes virulence by interfering with the chaperone activity of a barley small heat shock protein essential for defense and stress responses.

Trans-kingdom cross-talk: small RNAs on the move.

This review focuses on the mobility of small RNA (sRNA) molecules from the perspective of trans-kingdom gene silencing. Mobility of sRNA molecules within organisms is a well-known phenomenon, facilitating gene silencing between cells and tissues. sRNA signals are also transmitted between organisms of the same species and of different species. Remarkably, in recent years many examples of RNA-signal exchange have been described to occur between organisms of different kingdoms. These examples are predominantly found in interactions between hosts and their pathogens, parasites, and symbionts. However, they may only represent the tip of the iceberg, since the emerging picture suggests that organisms in biological niches commonly exchange RNA-silencing signals. In this case, we need to take this into account fully to understand how a given biological equilibrium is obtained. Despite many observations of trans-kingdom RNA signal transfer, several mechanistic aspects of these signals remain unknown. Such RNA signal transfer is already being exploited for practical purposes, though. Pathogen genes can be silenced by plant-produced sRNAs designed to affect these genes. This is also known as Host-Induced Genes Silencing (HIGS), and it has the potential to become an important disease-control method in the future.