Plant lysin motif extracellular proteins are required for arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal fungi (AMF) can form a mutually beneficial symbiotic relationship with most land plants. They are known to secrete lysin motif (LysM) effectors into host root cells for successful colonization. Intriguingly, plants secrete similar types of LysM proteins; however, their role in plant-microbe interactions is unknown. Here, we show that Medicago truncatula deploys LysM extracellular (LysMe) proteins to facilitate symbiosis with AMF. Promoter analyses demonstrated that three M. truncatula LysMe genes MtLysMe1/2/3, are expressed in arbuscule-containing cells and those adjacent to intercellular hyphae. Localization studies showed that these proteins are targeted to the periarbuscular space between the periarbuscular membrane and the fungal cell wall of the branched arbuscule. M. truncatula mutants in which MtLysMe2 was knocked out via CRISPR/Cas9-targeted mutagenesis exhibited a significant reduction in AMF colonization and arbuscule formation, whereas genetically complemented transgenic plants restored wild-type level AMF colonization. In addition, knocking out the ortholog of MtLysMe2 in tomato resulted in a similar defect in AMF colonization. In vitro binding affinity precipitation assays suggested binding of MtLysMe1/2/3 with chitin and chitosan, while microscale thermophoresis (MST) assays revealed weak binding of these proteins with chitooligosaccharides. Moreover, application of purified MtLysMe proteins to root segments could suppress chitooctaose (CO8)-induced reactive oxygen species production and expression of reporter genes of the immune response without impairing chitotetraose (CO4)-triggered symbiotic responses. Taken together, our results reveal that plants, like their fungal partners, also secrete LysM proteins to facilitate symbiosis establishment.

Transcription factors VviWRKY10 and VviWRKY30 co-regulate powdery mildew resistance in grapevine.

Grapevine (Vitis vinifera) is an economically important fruit crop worldwide. The widely cultivated grapevine is susceptible to powdery mildew caused by Erysiphe necator. In this study, we used CRISPR-Cas9 to simultaneously knock out VviWRKY10 and VviWRKY30 encoding two transcription factors reported to be implicated in defense regulation. We generated 53 wrky10 single mutant transgenic plants and 15 wrky10 wrky30 double mutant transgenic plants. In a 2-yr field evaluation of powdery mildew resistance, the wrky10 mutants showed strong resistance, while the wrky10 wrky30 double mutants showed moderate resistance. Further analyses revealed that salicylic acid (SA) and reactive oxygen species contents in the leaves of wrky10 and wrky10 wrky30 were substantially increased, as was the ethylene (ET) content in the leaves of wrky10. The results from dual luciferase reporter assays, electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP) assays demonstrated that VviWRKY10 could directly bind to the W-boxes in the promoter of SA-related defense genes and inhibit their transcription, supporting its role as a negative regulator of SA-dependent defense. By contrast, VviWRKY30 could directly bind to the W-boxes in the promoter of ET-related defense genes and promote their transcription, playing a positive role in ET production and ET-dependent defense. Moreover, VviWRKY10 and VviWRKY30 can bind to each other's promoters and mutually inhibit each other's transcription. Taken together, our results reveal a complex mechanism of regulation by VviWRKY10 and VviWRKY30 for activation of measured and balanced defense responses against powdery mildew in grapevine.

CRISPR-targeted mutagenesis of mitogen-activated protein kinase phosphatase 1 improves both immunity and yield in wheat.

Plants have evolved a sophisticated immunity system for specific detection of pathogens and rapid induction of measured defences. Over- or constitutive activation of defences would negatively affect plant growth and development. Hence, the plant immune system is under tight positive and negative regulation. MAP kinase phosphatase1 (MKP1) has been identified as a negative regulator of plant immunity in model plant Arabidopsis. However, the molecular mechanisms by which MKP1 regulates immune signalling in wheat (Triticum aestivum) are poorly understood. In this study, we investigated the role of TaMKP1 in wheat defence against two devastating fungal pathogens and determined its subcellular localization. We demonstrated that knock-down of TaMKP1 by CRISPR/Cas9 in wheat resulted in enhanced resistance to rust caused by Puccinia striiformis f. sp. tritici (Pst) and powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt), indicating that TaMKP1 negatively regulates disease resistance in wheat. Unexpectedly, while Tamkp1 mutant plants showed increased resistance to the two tested fungal pathogens they also had higher yield compared with wild-type control plants without infection. Our results suggested that TaMKP1 interacts directly with dephosphorylated and activated TaMPK3/4/6, and TaMPK4 interacts directly with TaPAL. Taken together, we demonstrated TaMKP1 exert negative modulating roles in the activation of TaMPK3/4/6, which are required for MAPK-mediated defence signalling. This facilitates our understanding of the important roles of MAP kinase phosphatases and MAPK cascades in plant immunity and production, and provides germplasm resources for breeding for high resistance and high yield.

Leaf abaxial immunity to powdery mildew in Arabidopsis is conferred by multiple defense mechanisms.

Powdery mildew fungi are obligate biotrophic pathogens that only invade plant epidermal cells. There are two epidermal surfaces in every plant leaf: the adaxial (upper) side and the abaxial (lower) side. While both leaf surfaces can be susceptible to adapted powdery mildew fungi in many plant species, there have been observations of leaf abaxial immunity in some plant species including Arabidopsis. The genetic basis of such leaf abaxial immunity remains unknown. In this study, we tested a series of Arabidopsis mutants defective in one or more known defense pathways with the adapted powdery mildew isolate Golovinomyces cichoracearum UCSC1. We found that leaf abaxial immunity was significantly compromised in mutants impaired for both the EDS1/PAD4- and PEN2/PEN3-dependent defenses. Consistently, expression of EDS1-yellow fluorescent protein and PEN2-green fluorescent protein fusions from their respective native promoters in the respective eds1-2 and pen2-1 mutant backgrounds was higher in the abaxial epidermal cells than in the adaxial epidermal cells. Altogether, our results indicate that leaf abaxial immunity against powdery mildew in Arabidopsis is at least partially due to enhanced EDS1/PAD4- and PEN2/PEN3-dependent defenses. Such transcriptionally pre-programmed defense mechanisms may underlie leaf abaxial immunity in other plant species such as hemp and may be exploited for engineering adaxial immunity against powdery mildew fungi in crop plants.

Wheat Pore-Forming Toxin-Like Protein Confers Broad-Spectrum Resistance to Fungal Pathogens in Arabidopsis.

Fusarium head blight (FHB), caused by the hemibiotrophic fungus Fusarium graminearum, is one of the major threats to global wheat productivity. A wheat pore-forming toxin-like (PFT) protein was previously reported to underlie Fhb1, the most widely used quantitative trait locus in FHB breeding programs worldwide. In the present work, wheat PFT was ectopically expressed in the model dicot plant Arabidopsis. Heterologous expression of wheat PFT in Arabidopsis provided a broad-spectrum quantitative resistance to fungal pathogens including F. graminearum, Colletotrichum higginsianum, Sclerotinia sclerotiorum, and Botrytis cinerea. However, there was no resistance to bacterial or oomycete pathogens Pseudomonas syringae and Phytophthora capsici, respectively in the transgenic Arabidopsis plants. To explore the reason for the resistance response to, exclusively, the fungal pathogens, purified PFT protein was hybridized to a glycan microarray having 300 different types of carbohydrate monomers and oligomers. It was found that PFT specifically hybridized with chitin monomer, N-acetyl glucosamine (GlcNAc), which is present in fungal cell walls but not in bacteria or oomycete species. This exclusive recognition of chitin may be responsible for the specificity of PFT-mediated resistance to fungal pathogens. Transfer of the atypical quantitative resistance of wheat PFT to a dicot system highlights its potential utility in designing broad-spectrum resistance in diverse host plants. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Golovinomyces cichoracearum effector-associated nuclear localization of RPW8.2 amplifies its expression to boost immunity in Arabidopsis.

Arabidopsis RESISTANCE TO POWDERY MILDEW 8.2 (RPW8.2) is specifically induced by the powdery mildew (PM) fungus (Golovinomyces cichoracearum) in the infected epidermal cells to activate immunity. However, the mechanism of RPW8.2-induction is not well understood. Here, we identify a G. cichoracearum effector that interacts with RPW8.2, named Gc-RPW8.2 interacting protein 1 (GcR8IP1), by a yeast two-hybrid screen of an Arabidopsis cDNA library. GcR8IP1 is physically associated with RPW8.2 with its REALLY INTERESTING NEW GENE finger domain that is essential and sufficient for the association. GcR8IP1 was secreted and translocated into the nucleus of host cell infected with PM. Association of GcR8IP1 with RPW8.2 led to an increase in RPW8.2 in the nucleus. In turn, the nucleus-localized RPW8.2 promoted the activity of the RPW8.2 promoter, resulting in transcriptional self-amplification of RPW8.2 to boost immunity at infection sites. Additionally, ectopic expression or host-induced gene silencing of GcR8IP1 supported its role as a virulence factor in PM. Altogether, our results reveal a mechanism of RPW8.2-dependent defense strengthening via altered partitioning of RPW8.2 and transcriptional self-amplification triggered by a PM fungal effector, which exemplifies an atypical form of effector-triggered immunity.

Integrated miRNA-mRNA analysis reveals candidate miRNA family regulating arbuscular mycorrhizal symbiosis of Poncirus trifoliata.

Over 70% land plants live in mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi, and maintenance of symbiosis requires transcriptional and post-transcriptional regulation. The former has been widely studied, whereas the latter mediated by symbiotic microRNAs (miRNAs) remains obscure, especially in woody plants. Here, we performed high-throughput sequencing of the perennial woody citrus plant Poncirus trifoliata and identified 3750 differentially expressed genes (DEGs) and 42 miRNAs (DEmiRs) upon AM fungal colonization. By analyzing cis-regulatory elements in the promoters of the DEGs, we predicted 329 key AM transcription factors (TFs). A miRNA-mRNA regulatory network was then constructed by integrating these data. Several candidate miRNA families of P. trifoliata were identified whose members target known symbiotic genes, such as miR167h-AMT2;3 and miR156e-EXO70I, or key TFs, such as miR164d-NAC and miR477a-GRAS, thus are involved in AM symbiotic processes of fungal colonization, arbuscule development, nutrient exchange and phytohormone signaling. Finally, analysis of selected miRNA family revealed that a miR159b conserved in mycorrhizal plant species and a Poncirus-specific miR477a regulate AM symbiosis. The role of miR477a was likely to target GRAS family gene RAD1 in citrus plants. Our results not only revealed that miRNA-mRNA network analysis, especially miRNA-TF analysis, is effective in identifying miRNA family regulating AM symbiosis, but also shed light on miRNA-mediated post-transcriptional regulation of AM symbiosis in woody citrus plants.

Genome Sequence Resource for Erysiphe necator NAFU1, a Grapevine Powdery Mildew Isolate Identified in Shaanxi Province of China.

Erysiphe necator is an economically important biotrophic fungal pathogen responsible for powdery mildew disease on grapevine. Currently, genome sequences are available for only a few E. necator isolates from the United States. Based on the combination of Nanopore and Illumina sequencing technologies, we present here the complete genome assembly for an isolate of E. necator, NAFU1, identified in China. We acquired a total of 15.93 Gb of raw reads. These reads were processed into a 61.12-Mb genome assembly containing 73 contigs with an N50 of 2.06 Mb and a maximum length of 6.05 Mb. Combining the results of three gene-prediction modules (i.e., an evidence-based gene modeler [EVidenceModeler], an ab initio gene modeler, and a homology-based gene modeler), we predicted 7,235 protein-coding genes in the assembled genome of E. necator NAFU1. This information will facilitate studies of genome evolution and pathogenicity mechanisms of E. necator and other powdery mildew species through comparative genome sequence analysis and other molecular genetic tools.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

AtSTP8, an endoplasmic reticulum-localised monosaccharide transporter from Arabidopsis, is recruited to the extrahaustorial membrane during powdery mildew infection.

Biotrophic pathogens are believed to strategically manipulate sugar transport in host cells to enhance their access to carbohydrates. However, mechanisms of sugar translocation from host cells to biotrophic fungi such as powdery mildew across the plant-haustorium interface remain poorly understood. To investigate this question, systematic subcellular localisation analysis was performed for all the 14 members of the monosaccharide sugar transporter protein (STP) family in Arabidopsis thaliana. The best candidate AtSTP8 was further characterised for its transport properties in Saccharomyces cerevisiae and potential role in powdery mildew infection by gene ablation and overexpression in Arabidopsis. Our results showed that AtSTP8 was mainly localised to the endoplasmic reticulum (ER) and appeared to be recruited to the host-derived extrahaustorial membrane (EHM) induced by powdery mildew. Functional complementation assays in S. cerevisiae suggested that AtSTP8 can transport a broad spectrum of hexose substrates. Moreover, transgenic Arabidopsis plants overexpressing AtSTP8 showed increased hexose concentration in leaf tissues and enhanced susceptibility to powdery mildew. Our data suggested that the ER-localised sugar transporter AtSTP8 may be recruited to the EHM where it may be involved in sugar acquisition by haustoria of powdery mildew from host cells in Arabidopsis.

Overexpression of two CDPKs from wild Chinese grapevine enhances powdery mildew resistance in Vitis vinifera and Arabidopsis.

Calcium-dependent protein kinases (CDPKs) play vital roles in metabolic regulations and stimuli responses in plants. However, little is known about their function in grapevine. Here, we report that VpCDPK9 and VpCDPK13, two paralogous CDPKs from Vitis pseudoreticulata accession Baihe-35-1, appear to positively regulate powdery mildew resistance. The transcription of them in leaves of 'Baihe-35-1' were differentially induced upon powdery mildew infection. Overexpression of VpCDPK9-YFP or VpCDPK13-YFP in the V. vinifera susceptible cultivar Thompson Seedless resulted in enhanced resistance to powdery mildew (YFP, yellow fluorescent protein). This might be due to elevation of SA and ethylene production, and excess accumulation of H2 O2 and callose in penetrated epidermal cells and/or the mesophyll cells underneath. Ectopic expression of VpCDPK9-YFP in Arabidopsis resulted in varied degrees of reduced stature, pre-mature senescence and enhanced powdery mildew resistance. However, these phenotypes were abolished in VpCDPK9-YFP transgenic lines impaired in SA signaling (pad4sid2) or ethylene signaling (ein2). Moreover, both of VpCDPK9 and VpCDPK13 were found to interact with and potentially phosphorylate VpMAPK3, VpMAPK6, VpACS1 and VpACS2 in vivo (ACS, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase; MAPK, mitogen-activated protein kinase). These results suggest that VpCDPK9 and VpCDPK13 contribute to powdery mildew resistance via positively regulating SA and ethylene signaling in grapevine.

RPW8.1 enhances the ethylene-signaling pathway to feedback-attenuate its mediated cell death and disease resistance in Arabidopsis.

The Arabidopsis RESISTANCE TO POWDERY MILDEW 8.1 (RPW8.1) activates confined cell death and defense against different pathogens. However, the underlying regulatory mechanisms still remain elusive. Here, we show that RPW8.1 activates ethylene signaling that, in turn, negatively regulates RPW8.1 expression. RPW8.1 binds and stabilizes 1-aminocyclopropane-1-carboxylate oxidase 4 (ACO4), which may in part explain increased ethylene production and signaling in RPW8.1-expressing plants. In return, ACO4 and other key components of ethylene signaling negatively regulate RPW8.1-mediated cell death and disease resistance via suppressing RPW8.1 expression. Loss of function in ACO4, EIN2, EIN3 EIL1, ERF6, ERF016 or ORA59 increases RPW8.1-mediated cell death and defense response. By contrast, overexpression of EIN3 abolishes or significantly compromises RPW8.1-mediated cell death and disease resistance. Furthermore, ERF6, ERF016 and ORA59 appear to act as trans-repressors of RPW8.1, with OAR59 being able to directly bind to the RPW8.1 promoter. Taken together, our results have revealed a feedback regulatory circuit connecting RPW8.1 and the ethylene-signaling pathway, in which RPW8.1 enhances ethylene signaling, and the latter, in return, negatively regulates RPW8.1-mediated cell death and defense response via suppressing RPW8.1 expression to attenuate its defense activity.

A High-Quality Genome Resource of Botrytis fragariae, a New and Rapidly Spreading Fungal Pathogen Causing Strawberry Gray Mold in the United States.

Although Botrytis fragariae was only recently identified as a new Botrytis species that causes strawberry gray mold, its prevalence extends to many states of the eastern United States. Compared with B. cinerea, which is known to be the causal agent of gray mold on many crop plants including strawberry, B. fragariae appears to have specifically adapted to strawberry and exhibits distinct fungicide sensitivity. This is the first presentation of a high-quality genome assembly of B. fragariae with gene annotation based on sequence homology and deep transcriptome data. The genome sequence information from B. fragariae is expected to help reveal genomic features underlying its host specialization and evolution of distinct fungicide resistance and other novel pathogenicity mechanisms.

In Silico Identification of the Full Complement of Subtilase-Encoding Genes and Characterization of the Role of TaSBT1.7 in Resistance Against Stripe Rust in Wheat.

Plant subtilases (SBTs) or subtilisin-like proteases comprise a very diverse family of serine peptidases that participates in a broad spectrum of biological functions. Despite increasing evidence for roles of SBTs in plant immunity in recent years, little is known about wheat (Triticum aestivum) SBTs (TaSBTs). Here, we identified 255 TaSBT genes from bread wheat using the latest version 2.0 of the reference genome sequence. The SBT family can be grouped into five clades, from TaSBT1 to TaSBT5, based on a phylogenetic tree constructed with deduced protein sequences. In silico protein-domain analysis revealed the existence of considerable sequence diversification of the TaSBT family which, together with the local clustered gene distribution, suggests that TaSBT genes have undergone extensive functional diversification. Among those TaSBT genes whose expression was altered by biotic factors, TaSBT1.7 was found to be induced in wheat leaves by chitin and flg22 elicitors, as well as six examined pathogens, implying a role for TaSBT1.7 in plant defense. Transient overexpression of TaSBT1.7 in Nicotiana benthamiana leaves resulted in necrotic cell death. Moreover, knocking down TaSBT1.7 in wheat using barley stripe mosaic virus-induced gene silencing compromised the hypersensitive response and resistance against Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust. Taken together, this study defined the full complement of wheat SBT genes and provided evidence for a positive role of one particular member, TaSBT1.7, in the incompatible interaction between wheat and a stripe rust pathogen.

First Report of Xanthomonas fragariae strain YL19 causing crown infection pockets in strawberry in Liaoning Province, China.

Strawberry (Fragaria × ananassa Duch.) is an important fruit crop in China. Typical crown infection pockets symptoms were observed on the infected strawberry in Liaoning province, China (121°60'E, 38°90'N) in the autumn of 2017. The disease incidence was estimated to be around 5 to 10 %, but could reach 30 to 40 % in some heavily affected plastic tunnels. The infected plants early displayed water-soaked symptoms on the abaxial leaf surface and subsequently developed reddish-brown shaped stripes and coalesced lesions on the adaxial leaf surface around the main veins (Fig. 1-A, 1-B). Several variable-size (0.3-0.8 mm in diameter) pockets were observed inside the crown tissues after dissection (Fig. 1-C). The diseased plants rarely reached fruiting and were easily broken between the crown tissue and the stem, and would eventually die. To identify the causal agent of this disease, the several surface-disinfested infected main veins and crown tissues were individually ground in sterile water and plated on sucrose peptone agar(SPA) medium (Hayward 1960) with 10-fold serial dilutions and incubated at 25℃. A number of yellow colonies grew on the medium at the 10-4 dilution 7 days after plating (Fig. 1-D) in all specimens. The colonies were aerobic, yellow, viscous, smooth, and gram-negative, which is a typical characteristic of Xanthomonas. To confirm identity of the causal bacteria, 18 colonies selected randomly were subjected to polymerase chain reactions (PCR) for the amplification of the cpn60 (Sahin et al. 2010), gyrB, rpoD, and fyuA (C Manceau et al. 2011), respectively. The results showed that the 18 colonies are identical. The cpn60, gyrB, rpoD, and fyuA sequences of this isolate were deposited in GenBank with accession numbers MT513132.1, MW233896, MW233897, and MW233895, respectively. BLAST searches with sequences of this isolate cpn60, gyrB, rpoD, and fyuA revealed 97.7%, 96.4%, 97.8%, and 97.3% similarity with the corresponding sequences of X. fragariae strain NBC2815 (LT853880.1), respectively. The resulting concatenated data set of cpn60-gyrB-rpoD-fyuA was used to build a Multilocus Sequence Analysis (MLSA) by maximum likelihood criteria (Fig. 2). The cpn60-gyrB-rpoD-fyuA sequences of the isolate from Liaoning clustered in the clade containing the type strain of X. fragariae NBC2815, indicating that it belongs to X. fragariae. Thus, the bacterial strain from Liaoning was designated as X. fragariae strain YL19. To fulfill Koch's postulates, the base of leaf petioles of disease-free strawberry plants were syringe-infiltrated inoculated with bacterial suspension (2×108 CFU) prepared from colonies of X. fragariae YL19 washed from SPA plates. The inoculated and control (treated with sterile water) were placed in a chamber (25/20℃day/night,≥90% relative humidity(RH), 12/12 h photoperiod) for three months. After one month, water-soaked symptoms were observed in the crown tissues of all X. fragariae YL19-inoculated plants. Two months after inoculation, a significant crown pocket similarly to initial symptoms observed in the field was developed on all inoculated plants. No symptoms were observed in the control plants. The bacteria were re-isolated from the symptomatic leaves, petioles and crowns, and confirmed as X. fragariae YL19 by the above mentioned morphological and molecular analyses. Pathogenicity tests were conducted three times and the same results were obtained. It was reported that X. fragariae usually causes angular leaf spot, a serious bacterial disease in many strawberry production regions worldwide. The typical symptoms of angular leaf spot caused by X. fragariae include reddish-brown, irregular spots on the upper leaf surface, water-soaked lesions developed along leaf veins. Although angular leaf spot caused by X. fragariae has been reported in Tianjin and Taiwan province, China (Wang et al. 2017; Wu et al. 2020), there is no report about the symptoms that infection pockets on crowns caused by X. fragariae strain YL19 as described above. This result indicated that YL19 is different from the other two X. fragariae strains reported in China or the disease caused by YL19 could be a severe case of angular leaf spot and vascular decline or collapse in strawberry (Bradbury, 1977). . To the best of our knowledge, these results showed a previously unreported new strain YL19 of X. fragariae is the causal agent of crown infecton pocket in strawberry in China, it may lead to serious losses to the local strawberry industry. This report will assist in developing management measures for this disease promptly.

ANNEXIN 8 negatively regulates RPW8.1-mediated cell death and disease resistance in Arabidopsis.

Study on the regulation of broad-spectrum resistance is an active area in plant biology. RESISTANCE TO POWDERY MILDEW 8.1 (RPW8.1) is one of a few broad-spectrum resistance genes triggering the hypersensitive response (HR) to restrict multiple pathogenic infections. To address the question how RPW8.1 signaling is regulated, we performed a genetic screen and tried to identify mutations enhancing RPW8.1-mediated HR. Here, we provided evidence to connect an annexin protein with RPW8.1-mediated resistance in Arabidopsis against powdery mildew. We isolated and characterized Arabidopsis b7-6 mutant. A point mutation in b7-6 at the At5g12380 locus resulted in an amino acid substitution in ANNEXIN 8 (AtANN8). Loss-of-function or RNA-silencing of AtANN8 led to enhanced expression of RPW8.1, RPW8.1-dependent necrotic lesions in leaves, and defense against powdery mildew. Conversely, over-expression of AtANN8 compromised RPW8.1-mediated disease resistance and cell death. Interestingly, the mutation in AtANN8 enhanced RPW8.1-triggered H2 O2 . In addition, mutation in AtANN8 led to hypersensitivity to salt stress. Together, our data indicate that AtANN8 is involved in multiple stress signaling pathways and negatively regulates RPW8.1-mediated resistance against powdery mildew and cell death, thus linking ANNEXIN's function with plant immunity.

Multiple intramolecular trafficking signals in RESISTANCE TO POWDERY MILDEW 8.2 are engaged in activation of cell death and defense.

The extrahaustorial membrane (EHM) is a host-derived interfacial membrane encasing the haustorium of powdery mildew fungi. Arabidopsis thaliana RESISTANCE TO POWDERY MILDEW 8.2 (RPW8.2) is specifically targeted to the EHM via two EHM-targeting signals. Here, we demonstrate that proper coordination between the trafficking forces engaged via the EHM-targeting signals and the nuclear localization signals (NLSs), as well as the nuclear export signals (NESs), in RPW8.2 is critical for the activation of cell death and defense. We show that in the absence of pathogens, RPW8.2 is partitioned between the cytoplasm and the nucleus, and turned over via both the 26S proteasome- and the vacuole-dependent pathways. Enhanced cytoplasmic localization of RPW8.2 by tagging it with a NES led to lethal cell death. By contrast, enhanced nuclear localization of RPW8.2 by adding an NLS to it resulted in resistance to powdery mildew. Whereas expression of the NES-containing C-terminal domain of RPW8.2 in the cytoplasm is sufficient to trigger cell death, no such cell death-inducing activity is found with RPW8.2 variants that contain the two EHM-targeting signals along with the NES-containing C-terminal domain. In addition, we present evidence for the involvement of a leaf senescence pathway in RPW8.2-mediated cell death and defense. Taken together, our data suggest that RPW8.2 is subject to adjustment by distinct and perhaps coordinated mechanisms for its localization and function via interaction with the multiple intramolecular trafficking signals, which should provide further insights into RPW8.2-activated, EHM-focused resistance against powdery mildew.

Sequencing and analysis of gerbera daisy leaf transcriptomes reveal disease resistance and susceptibility genes differentially expressed and associated with powdery mildew resistance.

BACKGROUND: RNA sequencing has been widely used to profile genome-wide gene expression and identify candidate genes controlling disease resistance and other important traits in plants. Gerbera daisy is one of the most important flowers in the global floricultural trade, and powdery mildew (PM) is the most important disease of gerbera. Genetic improvement of gerbera PM resistance has become a crucial goal in gerbera breeding. A better understanding of the genetic control of gerbera resistance to PM can expedite the development of PM-resistant cultivars. RESULTS: The objectives of this study were to identify gerbera genotypes with contrasting phenotypes in PM resistance and sequence and analyze their leaf transcriptomes to identify disease resistance and susceptibility genes differentially expressed and associated with PM resistance. An additional objective was to identify SNPs and SSRs for use in future genetic studies. We identified two gerbera genotypes, UFGE 4033 and 06-245-03, that were resistant and susceptible to PM, respectively. De novo assembly of their leaf transcriptomes using four complementary pipelines resulted in 145,348 transcripts with a N50 of 1124 bp, of which 67,312 transcripts contained open reading frames and 48,268 were expressed in both genotypes. A total of 494 transcripts were likely involved in disease resistance, and 17 and 24 transcripts were up- and down-regulated, respectively, in UFGE 4033 compared to 06-245-03. These gerbera disease resistance transcripts were most similar to the NBS-LRR class of plant resistance genes conferring resistance to various pathogens in plants. Four disease susceptibility transcripts (MLO-like) were expressed only or highly expressed in 06-245-03, offering excellent candidate targets for gene editing for PM resistance in gerbera. A total of 449,897 SNPs and 19,393 SSRs were revealed in the gerbera transcriptomes, which can be a valuable resource for developing new molecular markers. CONCLUSION: This study represents the first transcriptomic analysis of gerbera PM resistance, a highly important yet complex trait in a globally important floral crop. The differentially expressed disease resistance and susceptibility transcripts identified provide excellent targets for development of molecular markers and genetic maps, cloning of disease resistance genes, or targeted mutagenesis of disease susceptibility genes for PM resistance in gerbera.

A Medicago truncatula SWEET transporter implicated in arbuscule maintenance during arbuscular mycorrhizal symbiosis.

Plants form a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi, which facilitates the acquisition of scarce minerals from the soil. In return, the host plants provide sugars and lipids to its fungal partner. However, the mechanism by which the AM fungi obtain sugars from the plant has remained elusive. In this study we investigated the role of potential SWEET family sugar exporters in AM symbiosis in Medicago truncatula. We show that M. truncatula SWEET1b transporter is strongly upregulated in arbuscule-containing cells compared to roots and localizes to the peri-arbuscular membrane, across which nutrient exchange takes place. Heterologous expression of MtSWEET1b in a yeast hexose transport mutant showed that it mainly transports glucose. Overexpression of MtSWEET1b in M. truncatula roots promoted the growth of intraradical mycelium during AM symbiosis. Surprisingly, two independent Mtsweet1b mutants, which are predicted to produce truncated protein variants impaired in glucose transport, exhibited no significant defects in AM symbiosis. However, arbuscule-specific overexpression of MtSWEET1bY57A/G58D , which are considered to act in a dominant-negative manner, resulted in enhanced collapse of arbuscules. Taken together, our results reveal a (redundant) role for MtSWEET1b in the transport of glucose across the peri-arbuscular membrane to maintain arbuscules for a healthy mutually beneficial symbiosis.

Dual and Opposing Roles of Xanthine Dehydrogenase in Defense-Associated Reactive Oxygen Species Metabolism in Arabidopsis.

While plants produce reactive oxygen species (ROS) for stress signaling and pathogen defense, they need to remove excessive ROS induced during stress responses in order to minimize oxidative damage. How can plants fine-tune this balance and meet such conflicting needs? Here, we show that XANTHINE DEHYDROGENASE1 (XDH1) in Arabidopsis thaliana appears to play spatially opposite roles to serve this purpose. Through a large-scale genetic screen, we identified three missense mutations in XDH1 that impair XDH1's enzymatic functions and consequently affect the powdery mildew resistance mediated by RESISTANCE TO POWDERY MILDEW8 (RPW8) in epidermal cells and formation of xanthine-enriched autofluorescent objects in mesophyll cells. Further analyses revealed that in leaf epidermal cells, XDH1 likely functions as an oxidase, along with the NADPH oxidases RbohD and RbohF, to generate superoxide, which is dismutated into H2O2 The resulting enrichment of H2O2 in the fungal haustorial complex within infected epidermal cells helps to constrain the haustorium, thereby contributing to RPW8-dependent and RPW8-independent powdery mildew resistance. By contrast, in leaf mesophyll cells, XDH1 carries out xanthine dehydrogenase activity to produce uric acid in local and systemic tissues to scavenge H2O2 from stressed chloroplasts, thereby protecting plants from stress-induced oxidative damage. Thus, XDH1 plays spatially specified dual and opposing roles in modulation of ROS metabolism during defense responses in Arabidopsis.

Comparative genome analyses reveal sequence features reflecting distinct modes of host-adaptation between dicot and monocot powdery mildew.

BACKGROUND: Powdery mildew (PM) is one of the most important and widespread plant diseases caused by biotrophic fungi. Notably, while monocot (grass) PM fungi exhibit high-level of host-specialization, many dicot PM fungi display a broad host range. To understand such distinct modes of host-adaptation, we sequenced the genomes of four dicot PM biotypes belonging to Golovinomyces cichoracearum or Oidium neolycopersici. RESULTS: We compared genomes of the four dicot PM together with those of Blumeria graminis f.sp. hordei (both DH14 and RACE1 isolates), B. graminis f.sp. tritici, and Erysiphe necator infectious on barley, wheat and grapevine, respectively. We found that despite having a similar gene number (6620-6961), the PM genomes vary from 120 to 222 Mb in size. This high-level of genome size variation is indicative of highly differential transposon activities in the PM genomes. While the total number of genes in any given PM genome is only about half of that in the genomes of closely related ascomycete fungi, most (~ 93%) of the ascomycete core genes (ACGs) can be found in the PM genomes. Yet, 186 ACGs were found absent in at least two of the eight PM genomes, of which 35 are missing in some dicot PM biotypes, but present in the three monocot PM genomes, indicating remarkable, independent and perhaps ongoing gene loss in different PM lineages. Consistent with this, we found that only 4192 (3819 singleton) genes are shared by all the eight PM genomes, the remaining genes are lineage- or biotype-specific. Strikingly, whereas the three monocot PM genomes possess up to 661 genes encoding candidate secreted effector proteins (CSEPs) with families containing up to 38 members, all the five dicot PM fungi have only 116-175 genes encoding CSEPs with limited gene amplification. CONCLUSIONS: Compared to monocot (grass) PM fungi, dicot PM fungi have a much smaller effectorome. This is consistent with their contrasting modes of host-adaption: while the monocot PM fungi show a high-level of host specialization, which may reflect an advanced host-pathogen arms race, the dicot PM fungi tend to practice polyphagy, which might have lessened selective pressure for escalating an with a particular host.