SDE19, a SEC-dependent effector from 'Candidatus Liberibacter asiaticus' suppresses plant immunity and targets Citrus sinensis Sec12 to interfere with vesicle trafficking.

Citrus huanglongbing (HLB), which is caused by the phloem-colonizing bacteria Candidatus Liberibacter asiaticus (CLas), poses a significant threat to citrus production worldwide. The pathogenicity mechanism of HLB remains poorly understood. SEC-dependent effectors (SDEs) have been suggested to play critical roles in the interaction between citrus and CLas. Here, we explored the function of CLIBASIA_05320 (SDE19), a core SDE from CLas, and its interaction with its host target. Our data revealed that SDE19 is expressed at higher level during infection of citrus than that during infection of the Asian citrus psyllid. Subcellular localization assays showed that SDE19 is localized in the nucleus and cytoplasm and is capable of moving from cell to cell in Nicotiana benthamiana. To investigate whether SDE19 facilitates pathogen infection, we generated transgenic Arabidopsis thaliana and citrus plants overexpressing SDE19. Transgenic A. thaliana and citrus plants were more susceptible to Pseudomonas syringae pv. tomato (Pst) and Xanthomonas citri subsp. citri (Xcc), respectively. In addition, RNA-seq analysis demonstrated that overexpression of SDE19 resulted in a reprogramming of expression of genes related to biotic stimulus responses. SDE19 interacts with Citrus sinensis Sec12, a guanine nucleotide exchange factor responsible for the assembly of plant COPII (coat protein II)-coated vesicles, which mediate vesicle trafficking from the ER to the Golgi. SDE19 colocalizes with Sec12 in the ER by binding to its N-terminal catalytic region, affecting the stability of Sec12 through the 26S proteasome. This interaction hinders the secretion of apoplastic defense-related proteins such as PR1, P69B, GmGIP1, and RCR3. Furthermore, the secretion of PR1 and callose deposition is decreased in SDE19-transgenic A. thaliana. Taken together, SDE19 is a novel virulent SDE secreted by CLas that interacts with Sec12 to disrupt vesicle trafficking, inhibit defense-related proteins secretion, and promote bacterial infection. This study sheds light on how CLas manipulates the host vesicle trafficking pathway to suppress the secretion of defense-related proteins and interfere with plant immunity.

OrangeExpDB: an integrative gene expression database for Citrus spp.

BACKGROUND: Citrus is a major fruit crop, and RNA-sequencing (RNA-seq) data can be utilized to investigate its gene functions, heredity, evolution, development, and the detection of genes linked to essential traits or resistance to pathogens. However, it is challenging to use the public RNA-seq datasets for researchers without bioinformatics training, and expertise. RESULTS: OrangeExpDB is a web-based database that integrates transcriptome data of various Citrus spp., including C. limon (L.) Burm., C. maxima (Burm.) Merr., C. reticulata Blanco, C. sinensis (L.) Osbeck, and Poncirus trifoliata (L.) Raf., downloaded from the NCBI SRA database. It features a blast tool for browsing and searching, enabling quick download of expression matrices for different transcriptome samples. Expression of genes of interest can be easily generated by searching gene IDs or sequence similarity. Expression data in text format can be downloaded and presented as a heatmap, with additional sample information provided at the bottom of the webpage. CONCLUSIONS: Researchers can utilize OrangeExpDB to facilitate functional genomic analysis and identify key candidate genes, leveraging publicly available citrus RNA-seq datasets. OrangeExpDB can be accessed at http://www.orangeexpdb.com/ .

Phytophthora infestans RXLR effector PITG20303 targets a potato MKK1 protein to suppress plant immunity.

Pathogens secret a plethora of effectors into the host cell to modulate plant immunity. Analysing the role of effectors in altering the function of their host target proteins will reveal critical components of the plant immune system. Here we show that Phytophthora infestans RXLR effector PITG20303, a virulent variant of AVRblb2 (PITG20300) that escapes recognition by the resistance protein Rpi-blb2, suppresses PAMP-triggered immunity (PTI) and promotes pathogen colonization by targeting and stabilizing a potato MAPK cascade protein, StMKK1. Both PITG20300 and PITG20303 target StMKK1, as confirmed by multiple in vivo and in vitro assays, and StMKK1 was shown to be a negative regulator of plant immunity, as determined by overexpression and gene silencing. StMKK1 is a negative regulator of plant PTI, and the kinase activities of StMKK1 are required for its suppression of PTI and effector interaction. PITG20303 depends partially on MKK1, PITG20300 does not depend on MKK1 for suppression of PTI-induced reactive oxygen species burst, while the full virulence activities of nuclear targeted PITG20303 and PITG20300 are dependent on MKK1. Our results show that PITG20303 and PITG20300 target and stabilize the plant MAPK cascade signalling protein StMKK1 to negatively regulate plant PTI response.

The novel peptide NbPPI1 identified from Nicotiana benthamiana triggers immune responses and enhances resistance against Phytophthora pathogens.

In plants, recognition of small secreted peptides, such as damage/danger-associated molecular patterns (DAMPs), regulates diverse processes, including stress and immune responses. Here, we identified an SGPS (Ser-Gly-Pro-Ser) motif-containing peptide, Nicotiana tabacum NtPROPPI, and its two homologs in Nicotiana benthamiana, NbPROPPI1 and NbPROPPI2. Phytophthora parasitica infection and salicylic acid (SA) treatment induced NbPROPPI1/2 expression. Moreover, SignalP predicted that the 89-amino acid NtPROPPI includes a 24-amino acid N-terminal signal peptide and NbPROPPI1/2-GFP fusion proteins were mainly localized to the periplasm. Transient expression of NbPROPPI1/2 inhibited P. parasitica colonization, and NbPROPPI1/2 knockdown rendered plants more susceptible to P. parasitica. An eight-amino-acid segment in the NbPROPPI1 C-terminus was essential for its immune function and a synthetic 20-residue peptide, NbPPI1, derived from the C-terminus of NbPROPPI1 provoked significant immune responses in N. benthamiana. These responses led to enhanced accumulation of reactive oxygen species, activation of mitogen-activated protein kinases, and up-regulation of the defense genes Flg22-induced receptor-like kinase (FRK) and WRKY DNA-binding protein 33 (WRKY33). The NbPPI1-induced defense responses require Brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1). These results suggest that NbPPI1 functions as a DAMP in N. benthamiana; this novel DAMP provides a potentially useful target for improving plant resistance to Pytophthora pathogens.

Negative regulators of plant immunity derived from cinnamyl alcohol dehydrogenases are targeted by multiple Phytophthora Avr3a-like effectors.

Oomycete pathogens secrete numerous effectors to manipulate host immunity. While some effectors share a conserved structural fold, it remains unclear if any have conserved host targets. Avr3a-like family effectors, which are related to Phytophthora infestans effector PiAvr3a and are widely distributed across diverse clades of Phytophthora species, were used to study this question. By using yeast-two-hybrid, bimolecular fluorescence complementation and co-immunoprecipitation assays, we identified members of the plant cinnamyl alcohol dehydrogenase 7 (CAD7) subfamily as targets of multiple Avr3a-like effectors from Phytophthora pathogens. The CAD7 subfamily has expanded in plant genomes but lost the lignin biosynthetic activity of canonical CAD subfamilies. In turn, we identified CAD7s as negative regulators of plant immunity that are induced by Phytophthora infection. Moreover, AtCAD7 was stabilized by Avr3a-like effectors and involved in suppression of pathogen-associated molecular pattern-triggered immunity, including callose deposition, reactive oxygen species burst and WRKY33 expression. Our results reveal CAD7 subfamily proteins as negative regulators of plant immunity that are exploited by multiple Avr3a-like effectors to promote infection in different host plants.

Analysis of huanglongbing-associated RNA-seq data reveals disturbances in biological processes within Citrus spp. triggered by Candidatus Liberibacter asiaticus infection.

Introduction: Huanglongbing (HLB), a disease that's ubiquitous worldwide, wreaks havoc on the citrus industry. The primary culprit of HLB is the gram-negative bacterium Candidatus Liberibacter asiaticus (CLas) that infects the phloem, but its damaging mechanism is yet to be fully understood. Methods and results: In this study, a multitude of tools including weighted correlation network analysis (WGCNA), protein-protein interaction (PPI) network analysis and gene expression profiling are employed to unravel the intricacies of its pathogenesis. The investigation pinpoints various central genes, such as the ethylene-responsive transcription factor 9 (ERF9) and thioredoxin reductase 1 (TrxR1), that are associated with CLas invasion and resultant disturbances in numerous biological operations. Additionally, the study uncovers a range of responses through the detection of differential expressed genes (DEGs) across different experiments. The discovery of core DEGs leads to the identification of pivotal genes such as the sieve element occlusion (SEO) and the wall-associated receptor kinase-like 15 (WAKL15). PPI network analysis highlights potential vital proteins, while GO and KEGG pathway enrichment analysis illustrate a significant impact on multiple defensive and metabolic pathways. Gene set enrichment analysis (GSEA) indicates significant alterations in biological processes such as leaf senescence and response to biotic stimuli. Discussion: This all-encompassing approach extends valuable understanding into the pathogenesis of CLas, potentially aiding future research and therapeutic strategies for HLB.

Transcriptome analysis of Citrus sinensis reveals potential responsive events triggered by Candidatus Liberibacter asiaticus.

Citrus Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), is a devastating immune-mediated disorder that has a detrimental effect on the citrus industry, with the distinguishing feature being an eruption of reactive oxygen species (ROS). This study explored the alterations in antioxidant enzyme activity, transcriptome, and RNA editing events of organelles in C. sinensis during CLas infection. Results indicated that there were fluctuations in the performance of antioxidant enzymes, such as ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), peroxidase (POD), and superoxide dismutase (SOD), in plants affected by HLB. Transcriptome analysis revealed 3604 genes with altered expression patterns between CLas-infected and healthy samples, including those associated with photosynthesis, biotic interactions, and phytohormones. Samples infected with CLas showed a decrease in the expression of most genes associated with photosynthesis and gibberellin metabolism. It was discovered that RNA editing frequency and the expression level of various genes in the chloroplast and mitochondrion genomes were affected by CLas infection. Our findings provide insights into the inhibition of photosynthesis, gibberellin metabolism, and antioxidant enzymes during CLas infection in C. sinensis.

Genome-wide characterization of heavy metal-associated isoprenylated plant protein gene family from Citrus sinensis in response to huanglongbing.

Introduction: Heavy metal-associated isoprenylated plant proteins (HIPPs) play vital roles in maintaining heavy metal balance and responding to both biotic and abiotic stresses in vascular plants. However, the role of HIPPs in the response to Huanglongbing (HLB), a harmful disease of citrus caused by the phloem-colonizing bacterium Candidatus Liberibacter asiaticus (CLas), has not been examined. Methods and results: In this study, a total of 26 HIPP genes were identified in Citrus sinensis, and they were grouped into 5 clades. The CsHIPP genes are distributed on 8 chromosomes and exhibited considerable synteny with HIPPs found in Arabidopsis thaliana. Additionally, we analyzed the gene structure, conserved motifs and domains of the CsHIPPs. Various cis-acting elements related to plant hormones and stress responses were identified in the promoters of CsHIPPs. Public transcriptome data and RT-qPCR analysis showed that the expression level of CsHIPP03 was significantly reduced in samples infected by CLas and Xanthomonas citri ssp. citri (Xcc). Furthermore, silencing the homologous gene of CsHIPP03 in Nicotiana benthamiana increased the disease resistance of plants to bacteria. Discussion: Our results provide a basis for functional studies of HIPP gene family in C. sinensis, highlighting their functions in bacterial resistance, and improve our understanding to the susceptibility mechanism of HLB.

Synthesis and Properties of Room-Temperature Phosphorescent Liquid Crystal Copolymers with Linearly Polarized Luminescence Characteristic.

Room-temperature phosphorescent (RTP) liquid crystal materials have garnered considerable attention because of their significant applications in organic light emitting diodes, polarized light emitting materials, and so forth. How to efficiently synthesize pure organic RTP liquid crystals and regulate their performance is of great significance. In this article, we propose a simple and feasible method to synthesize RTP liquid crystals and manipulate their properties through copolymerization. We constructed RTP liquid crystal copolymers by copolymerizing a phosphorescent monomer bearing biphenyl mesogen with a phosphorescent monomer bearing a dibenzofuran chromophore. All the synthesized copolymers show a liquid crystal property because of the introduction of biphenyl mesogen. Meanwhile, by changing the composition of copolymers, it is possible to regulate their RTP performance, including luminescence color and lifetime. As the content of the PMDFM0C component in copolymers increases, the phosphorescence lifetime gradually increases. For poly(MDFM0C(0.46)-co-MBi18C(0.54)), the phosphorescence lifetime can reach 463.0 ms. Moreover, the phosphorescence color of the PMDFM0C component in copolymers changes with the copolymer composition, which can induce variable room-temperature phosphorescence. In addition, when oriented, liquid crystal copolymer films can emit linearly polarized fluorescence and linearly polarized phosphorescence. The linearly polarized phosphorescence dichroic ratio and polarization ratio values of the oriented poly(MDFM0C(0.46)-co-MBi18C(0.54)) film are 3.33 and 0.50, respectively.

Systematic analysis of the thioredoxin gene family in Citrus sinensis: identification, phylogenetic analysis, and gene expression patterns.

Thioredoxin (TRX) proteins play essential roles in reactive oxygen species scavenging in plants. We executed an exhaustive analysis of the TRX gene family in Citrus sinensis (CsTRXs), encompassing identification, phylogenetic analysis, detection of conserved motifs and domains, gene structure, cis-acting elements, gene expression trends, and subcellular localization analysis. Our findings established that a total of 22 CsTRXs with thioredoxin domains were identified in the genome of C. sinensis. Phylogenetic analysis indicated that CsTRXs were divided into six subclusters. Conserved motifs analysis of CsTRXs indicated a wide range of conserved motifs. A significant number of cis-acting elements associated with both abiotic and biotic stress responses, inclusive of numerous phytohormone-related elements, were detected in the promoter regions of CsTRXs. The expression levels of CsTRXs including CsTRXf1, CsTRXh1, CsTRXm1, CsTRXo3, CsTRXx2 and CsTRXy1 were observed to be reduced upon pathogen infection. Subcellular localization analysis found that CsTRXf1, CsTRXm1, CsTRXo3, CsTRXx2 and CsTRXy1 were predominantly localized in chloroplasts, whereas CsTRXh1 was distributed indiscriminately. This research yields integral data on CsTRXs, facilitating future efforts to decipher the gene functions of CsTRXs.

Mutations in PpAGO3 Lead to Enhanced Virulence of Phytophthora parasitica by Activation of 25-26 nt sRNA-Associated Effector Genes.

Oomycetes represent a unique group of plant pathogens that are destructive to a wide range of crops and natural ecosystems. Phytophthora species possess active small RNA (sRNA) silencing pathways, but little is known about the biological roles of sRNAs and associated factors in pathogenicity. Here we show that an AGO gene, PpAGO3, plays a major role in the regulation of effector genes hence the pathogenicity of Phytophthora parasitica. PpAGO3 was unique among five predicted AGO genes in P. parasitica, showing strong mycelium stage-specific expression. Using the CRISPR-Cas9 technology, we generated PpAGO3ΔRGG1-3 mutants that carried a deletion of 1, 2, or 3 copies of the N-terminal RGG motif (QRGGYD) but failed to obtain complete knockout mutants, which suggests its vital role in P. parasitica. These mutants showed increased pathogenicity on both Nicotiana benthamiana and Arabidopsis thaliana plants. Transcriptome and sRNA sequencing of PpAGO3ΔRGG1 and PpAGO3ΔRGG3 showed that these mutants were differentially accumulated with 25-26 nt sRNAs associated with 70 predicted cytoplasmic effector genes compared to the wild-type, of which 13 exhibited inverse correlation between gene expression and 25-26 nt sRNA accumulation. Transient overexpression of the upregulated RXLR effector genes, PPTG_01869 and PPTG_15425 identified in the mutants PpAGO3ΔRGG1 and PpAGO3ΔRGG3 , strongly enhanced N. benthamiana susceptibility to P. parasitica. Our results suggest that PpAGO3 functions together with 25-26 nt sRNAs to confer dynamic expression regulation of effector genes in P. parasitica, thereby contributing to infection and pathogenicity of the pathogen.

An RXLR effector secreted by Phytophthora parasitica is a virulence factor and triggers cell death in various plants.

RXLR effectors encoded by Phytophthora species play a central role in pathogen-plant interactions. An understanding of the biological functions of RXLR effectors is conducive to the illumination of the pathogenic mechanisms and the development of disease control strategies. However, the virulence function of Phytophthora parasitica RXLR effectors is poorly understood. Here, we describe the identification of a P. parasitica RXLR effector gene, PPTG00121 (PpE4), which is highly transcribed during the early stages of infection. Live cell imaging of P. parasitica transformants expressing a full-length PpE4 (E4FL)-mCherry protein indicated that PpE4 is secreted and accumulates around haustoria during plant infection. Silencing of PpE4 in P. parasitica resulted in significantly reduced virulence on Nicotiana benthamiana. Transient expression of PpE4 in N. benthamiana in turn restored the pathogenicity of the PpE4-silenced lines. Furthermore, the expression of PpE4 in both N. benthamiana and Arabidopsis thaliana consistently enhanced plant susceptibility to P. parasitica. These results indicate that PpE4 contributes to pathogen infection. Finally, heterologous expression experiments showed that PpE4 triggers non-specific cell death in a variety of plants, including tobacco, tomato, potato and A. thaliana. Virus-induced gene silencing assays revealed that PpE4-induced cell death is dependent on HSP90, NPK and SGT1, suggesting that PpE4 is recognized by the plant immune system. In conclusion, PpE4 is an important virulence RXLR effector of P. parasitica and recognized by a wide range of host plants.

Correction to: Identification of ß­carboline and canthinone alkaloids as anti­inflammatory agents but with different inhibitory profile on the expression of iNOS and COX­2 in lipopolysaccharide­activated RAW 264.7 macrophages.

The article Identification of ß­carboline and canthinone alkaloids as anti­inflammatory agents but with different inhibitory profile on the expression of iNOS and COX­2 in lipopolysaccharide­activated RAW 264.7 macrophages, written by Pan Liu, Huixiang Li, Ruiling Luan, Guiyan Huang, Yanan Liu, Mengdi Wang, Qiuli Chao, Liying Wang, Danna Li, Huaying Fan, Daquan Chen, Linyu Li, Keiichi Matsuzaki, Wei Li, Kazuo Koike, Feng Zhao, was originally published electronically on the publisher's internet portal (currently SpringerLink) on 15 October 2018 without open access.

Identification of ß-carboline and canthinone alkaloids as anti-inflammatory agents but with different inhibitory profile on the expression of iNOS and COX-2 in lipopolysaccharide-activated RAW 264.7 macrophages.

A compound library, which consists of 75 natural ß-carboline-type or canthinone-type alkaloids from Simaroubaceae plants and their chemical synthetic analogues, was screened for the anti-inflammatory activity by inhibition of the overproduction of inflammatory mediator nitric oxide (NO) in lipopolysaccharide (LPS)-activated RAW 264.7 macrophage cells. Six compounds, namely, benzalharman (23), kumujian (27), 1-ethyl-1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid (37), 1-acetophenone-1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid (42), cathin-6-one (46), and 9-methoxy-cathin-6-one (57), exhibited significant inhibitory activity on the overproduction of NO with good dose dependency. Further investigation demonstrated that all of the six compounds down-regulated the high expression of inducible nitric oxide synthase (iNOS) protein. Among them, two canthinone-type alkaloids (46 and 57) potently down-regulated cyclooxygenase-2 (COX-2) protein expression in a dose-dependent manner and also inhibited the overproduction of inflammatory mediator prostaglandin E2 (PGE2). However, the ß-carboline-type alkaloids (23, 27, 37, and 42) exhibited no obvious inhibition on the overproduction of PGE2 and the expression of COX-2 protein. The results suggested that ß-carboline-type alkaloids and canthinone-type alkaloids may exert an anti-inflammatory effect through different mechanism.

Conserved RXLR Effector Genes of Phytophthora infestans Expressed at the Early Stage of Potato Infection Are Suppressive to Host Defense.

Late blight has been the most devastating potato disease worldwide. The causal agent, Phytophthora infestans, is notorious for its capability to rapidly overcome host resistance. Changes in the expression pattern and the encoded protein sequences of effector genes in the pathogen are responsible for the loss of host resistance. Among numerous effector genes, the class of RXLR effector genes is well-known in mediating host genotype-specific resistance. We therefore performed deep sequencing of five genetically diverse P. infestans strains using in planta materials infected with zoospores (12 h post inoculation) and focused on the identification of RXLR effector genes that are conserved in coding sequences, are highly expressed in early stages of plant infection, and have defense suppression activities. In all, 245 RXLR effector genes were expressed in five transcriptomes, with 108 being co-expressed in all five strains, 47 of them comparatively highly expressed. Taking sequence polymorphism into consideration, 18 candidate core RXLR effectors that were conserved in sequence and with higher in planta expression levels were selected for further study. Agrobacterium tumefaciens-mediated transient expression of the selected effector genes in Nicotiana benthamiana and potato demonstrated their potential virulence function, as shown by suppression of PAMP-triggered immunity (PTI) or/and effector-triggered immunity (ETI). The identified collection of core RXLR effectors will be useful in the search for potential durable late blight resistance genes. Analysis of 10 known Avr RXLR genes revealed that the resistance genes R2, Rpi-blb2, Rpi-vnt1, Rpi-Smira1, and Rpi-Smira2 may be effective in potato cultivars. Analysis of 8 SFI (Suppressor of early Flg22-induced Immune response) RXLR effector genes showed that SFI2, SFI3, and SFI4 were highly expressed in all examined strains, suggesting their potentially important function in early stages of pathogen infection.

The protein disulfide isomerase 1 of Phytophthora parasitica (PpPDI1) is associated with the haustoria-like structures and contributes to plant infection.

Protein disulfide isomerase (PDI) is a ubiquitous and multifunction enzyme belonging to the thioredoxin (TRX) superfamily, which can reduce, oxidize, and catalyze dithiol-disulfide exchange reactions. Other than performing housekeeping functions in helping to maintain proteins in a more stable conformation, there is some evidence to indicate that PDI is involved in pathogen infection processes. In a high-throughput screening for necrosis-inducing factors by Agrobacterium tumefaciens-mediated transient expression assay, a typical PDI gene from Phytophthora parasitica (PpPDI1) was identified and confirmed to induce strong cell death in Nicotiana benthamiana leaves. PpPDI1 is conserved in eukaryotes but predicted to be a secreted protein. Deletion mutant analyses showed that the first CGHC motif in the active domain of PpPDI1 is essential for inducing cell death. Using P. parasitica transformation method, the silencing efficiency was found to be very low, suggesting that PpPDI1 is essential for the pathogen. Translational fusion to the enhanced green fluorescent protein (EGFP) in stable P. parasitica transformants showed that PpPDI1 is associated with haustoria-like structures during pathogen infection. Furthermore, the PpPDI1-EGFP-expressing transformants increase the number of haustoria-like structures and exhibit enhanced virulence to N. benthamiana. These results indicate that PpPDI1 might be a virulence factor of P. parasitica and contributes to plant infection.

Phenotypic and genetic characterization of resistance in Arabidopsis thaliana to the oomycete pathogen Phytophthora parasitica.

The interaction between Arabidopsis thaliana and the oomycete pathogen Phytophthora parasitica emerges as a model for exploring the molecular basis and evolution of recognition and host defense. Phenotypic variation and genetic analysis is essential to dissect the underlying mechanisms in plant-oomycete interaction. In this study, the reaction phenotypes of 28 A. thaliana accessions to P. parasitica strain Pp016 were examined using detached leaf infection assay. The results showed the presence of four distinct groups based on host response and disease development. Of all the accessions examined, Zurich (Zu-1) is highly resistant to P. parasitica. Microscopic characterization showed that rapid and severe hypersensitive response at the primary infection epidermal cells is associated with disease resistance. Furthermore, Zu-1 is resistant to a set of 20 diverse P. parasitica strains, which were collected from different host plants and exhibited differential specificities on a set of tobacco cultivars. However, Zu-1 is susceptible to P. parasitica when the root is inoculated, suggesting differential expression of associated resistance genes in the root and foliar tissues. Genetic analysis by crossing Zu-1 and the susceptible accession Landsberg (Ler) showed that the resistance in Zu-1 to P. parasitica is semi-dominant, as shown by infection assays of F1 progenies, and is likely conferred by a single locus, defined as RPPA1 (Zu-1) (for Resistance to P. parasitica 1), as shown by analysis of F2 segregating populations. By employing specific-locus amplified fragment sequencing (SLAF-seq) strategy to identify molecular markers potentially linked to the locus, the strongest associated region was determined to be located between 7.1 and 11.2 Mb in chromosome IV. The future cloning of RPPA1 (Zu-1) locus will facilitate improved understanding of plant broad-spectrum disease resistance to oomycete pathogens.