Multiple Forms of Resistance to the Phomopsis Stem Canker Pathogens Diaporthe helianthi and D. gulyae in Sunflower.

Phomopsis stem canker of cultivated sunflower (Helianthus annuus L.) can be caused by multiple necrotrophic fungi in the genus Diaporthe, with Diaporthe helianthi and D. gulyae being the most common causal agents in the United States. Infection begins at the leaf margins and proceeds primarily through the vasculature, progressing from the leaf through the petiole to the stem, resulting in formation of brown stem lesions centered around the petiole. Sunflower resistance to Phomopsis stem canker is quantitative and genetically complex. Due to the intricate disease process, resistance is possible at different stages of infection, and multiple forms of defense may contribute to the overall level of quantitative resistance. In this study, sunflower lines exhibiting field resistance to Phomopsis stem canker were evaluated for stem and leaf resistance to multiple isolates of D. helianthi and D. gulyae in greenhouse experiments, and responses to the two species were compared. Additionally, selected resistant and susceptible lines were evaluated for petiole transmission resistance to D. helianthi. Lines with distinct forms of resistance were identified, and results indicated that responses to stem inoculation were strongly correlated (Spearman's coefficient 0.598, P < 0.001) for the two fungal species, while leaf responses were not (Spearman's coefficient 0.396, P = 0.076). These results provide a basis for genetic dissection of distinct forms of sunflower resistance to Phomopsis stem canker and will facilitate combining different forms of resistance to potentially achieve durable control of this disease in sunflower hybrids.

Heritable differences in abundance of bacterial rhizosphere taxa are correlated with fungal necrotrophic pathogen resistance.

Host-microbe interactions are increasingly recognized as important drivers of organismal health, growth, longevity and community-scale ecological processes. However, less is known about how genetic variation affects hosts' associated microbiomes and downstream phenotypes. We demonstrate that sunflower (Helianthus annuus) harbours substantial, heritable variation in microbial communities under field conditions. We show that microbial communities co-vary with heritable variation in resistance to root infection caused by the necrotrophic pathogen Sclerotinia sclerotiorum and that plants grown in autoclaved soil showed almost complete elimination of pathogen resistance. Association mapping suggests at least 59 genetic locations with effects on both microbial relative abundance and Sclerotinia resistance. Although the genetic architecture appears quantitative, we have elucidated previously unexplained genetic variation for resistance to this pathogen. We identify new targets for plant breeding and demonstrate the potential for heritable microbial associations to play important roles in defence in natural and human-altered environments.

Genetic analysis of basal stalk rot resistance introgressed from wild Helianthus petiolaris into cultivated sunflower (Helianthus annuus L.) using an advanced backcross population.

Introduction: Sclerotinia sclerotiorum is a serious pathogen causing severe basal stalk rot (BSR) disease on cultivated sunflower (Helianthus annuus L.) that leads to significant yield losses due to insufficient resistance. The wild annual sunflower species H. petiolaris, commonly known as prairie sunflower is known for its resistance against this pathogen. Sunflower resistance to BSR is quantitative and determined by many genes with small effects on the resistance phenotype. The objective of this study was to identify loci governing BSR resistance derived from H. petiolaris using a quantitative trait loci (QTL) mapping approach. Methods: BSR resistance among lines of an advanced backcross population (AB-QTL) with 174 lines developed from a cross of inbred line HA 89 with H. petiolaris PI 435843 was determined in the field during 2017-2019, and in the greenhouse in 2019. AB-QTL lines and the HA 89 parent were genotyped using genotyping-by-sequencing and a genetic linkage map was developed spanning 997.51 cM and using 1,150 SNP markers mapped on 17 sunflower chromosomes. Results and discussion: Highly significant differences (p<0.001) for BSR response among AB-QTL lines were observed disease incidence (DI) in all field seasons, as well as disease rating (DR) and area under the disease progress curve (AUDPC) in the greenhouse with a moderately high broad-sense heritability (H 2) of 0.61 for the tested resistance parameters. A total of 14 QTL associated with BSR resistance were identified on nine chromosomes, each explaining a proportion of the phenotypic variation ranging from 3.5% to 28.1%. Of the 14 QTL, eight were detected for BSR resistance in the field and six were detected under greenhouse conditions. Alleles conferring increased BSR resistance were contributed by the H. petiolaris parent at 11 of the 14 QTL.

Population and genome-wide association studies of Sclerotinia sclerotiorum isolates collected from diverse host plants throughout the United States.

Introduction: Sclerotinia sclerotiorum is a necrotrophic fungal pathogen causing disease and economic loss on numerous crop plants. This fungus has a broad host range and can infect over 400 plant species, including important oilseed crops such as soybean, canola, and sunflower. S. sclerotiorum isolates vary in aggressiveness of lesion formation on plant tissues. However, the genetic basis for this variation remains to be determined. The aims of this study were to evaluate a diverse collection of S. sclerotiorum isolates collected from numerous hosts and U.S. states for aggressiveness of stem lesion formation on sunflower, to evaluate the population characteristics, and to identify loci associated with isolate aggressiveness using genome-wide association mapping. Methods: A total of 219 S. sclerotiorum isolates were evaluated for stem lesion formation on two sunflower inbred lines and genotyped using genotyping-by-sequencing. DNA markers were used to assess population differentiation across hosts, regions, and climatic conditions and to perform a genome-wide association study of isolate aggressiveness. Results and discussion: We observed a broad range of aggressiveness for lesion formation on sunflower stems, and only a moderate correlation between aggressiveness on the two lines. Population genetic evaluations revealed differentiation between populations from warmer climate regions compared to cooler regions. Finally, a genome-wide association study of isolate aggressiveness identified three loci significantly associated with aggressiveness on sunflower. Functional characterization of candidate genes at these loci will likely improve our understanding of the virulence strategies used by this pathogen to cause disease on a wide array of agriculturally important host plants.

Mutation and sequencing-based cloning and functional studies of a rust resistance gene in sunflower (Helianthus annuus).

Rust, caused by the fungus Puccinia helianthi Schwein., is one of the most devastating diseases of sunflower (Helianthus annuus L.), affecting global production. The rust R gene R11 in sunflower line HA-R9 shows broad-spectrum resistance to P. helianthi virulent races and was previously mapped to an interval on sunflower chromosome 13 encompassing three candidate genes annotated in the XRQr1.0 reference genome assembly. In the current study, we combined ethyl methane sulfonate (EMS) mutagenesis with targeted region capture and PacBio long-read sequencing to clone the R11 gene. Sequencing of a 60-kb region spanning the R11 locus from the R11 -HA-R9 rust-resistant line and three EMS-induced susceptible mutants facilitated the identification of R11 and definition of induced mutations. The R11 gene is predicted to have a single 3996-bp open reading frame and encodes a protein of 1331 amino acids with CC-NBS-LRR domains typical of genes conferring plant resistance to biotrophic pathogens. Point mutations identified in the R11 rust-susceptible mutants resulted in premature stop codons, consistent with loss of function leading to rust susceptibility. Additional functional studies using comparative RNA sequencing of the resistant line R11 -HA-R9 and R11 -susceptible mutants revealed substantial differences in gene expression patterns associated with R11 -mediated resistance at 7 days post-inoculation with rust, and uncovered the potential roles of terpenoid biosynthesis and metabolism in sunflower rust resistance.

A Quantitative Genetic Study of Sclerotinia Head Rot Resistance Introgressed from the Wild Perennial Helianthus maximiliani into Cultivated Sunflower (Helianthus annuus L.).

Sclerotinia head rot (HR), caused by Sclerotinia sclerotiorum, is an economically important disease of sunflower with known detrimental effects on yield and quality in humid climates worldwide. The objective of this study was to gain insight into the genetic architecture of HR resistance from a sunflower line HR21 harboring HR resistance introgressed from the wild perennial Helianthus maximiliani. An F2 population derived from the cross of HA 234 (susceptible-line)/HR21 (resistant-line) was evaluated for HR resistance at two locations during 2019−2020. Highly significant genetic variations (p < 0.001) were observed for HR disease incidence (DI) and disease severity (DS) in both individual and combined analyses. Broad sense heritability (H2) estimates across environments for DI and DS were 0.51 and 0.62, respectively. A high-density genetic map of 1420.287 cM was constructed with 6315 SNP/InDel markers developed using genotype-by-sequencing technology. A total of 16 genomic regions on eight sunflower chromosomes, 1, 2, 10, 12, 13, 14, 16 and 17 were associated with HR resistance, each explaining between 3.97 to 16.67% of the phenotypic variance for HR resistance. Eleven of these QTL had resistance alleles from the HR21 parent. Molecular markers flanking the QTL will facilitate marker-assisted selection breeding for HR resistance in sunflower.

Genomic Insights Into Sclerotinia Basal Stalk Rot Resistance Introgressed From Wild Helianthus praecox Into Cultivated Sunflower (Helianthus annuus L.).

Crop wild relatives of the cultivated sunflower (Helianthus annuus L.) are a valuable resource for its sustainable production. Helianthus praecox ssp. runyonii is a wild sunflower known for its resistance against diseases caused by the fungus, Sclerotinia sclerotiorum (Lib.) de Bary, which infects over 400 broadleaf hosts including many important food crops. The objective of this research was to dissect the Sclerotinia basal stalk rot (BSR) resistance introgressed from H. praecox ssp. runyonii into cultivated sunflower. An advanced backcross quantitative trait loci (AB-QTL) mapping population was developed from the cross of a H. praecox accession with cultivated sunflower lines. The AB-QTL population was evaluated for BSR resistance in the field during the summers of 2017-2018 and in the greenhouse in the spring of 2018. Highly significant genetic variations (p < 0.001) were observed for the BSR disease in the field and greenhouse with a moderately high broad-sense heritability (H 2) ranging from 0.66 to 0.73. Genotyping-by-sequencing approach was used to genotype the parents and the progeny lines of the AB-QTL population. A genetic linkage map spanning 1,802.95 cM was constructed using 1,755 single nucleotide polymorphism (SNP) markers mapped on 17 sunflower chromosomes. A total of 19 BSR resistance QTL were detected on nine sunflower chromosomes, each explaining 2.21%-16.99% of the phenotypic variance for resistance in the AB-QTL population. Sixteen of the 19 QTL had alleles conferring increased BSR resistance derived from the H. praecox parent. SNP markers flanking the identified QTL will facilitate marker-assisted breeding to combat the disease in sunflower.

Arabidopsis GOLD36/MVP1/ERMO3 Is Required for Powdery Mildew Penetration Resistance and Proper Targeting of the PEN3 Transporter.

The Arabidopsis PENETRATION 3 (PEN3) ATP binding cassette (ABC) transporter contributes to penetration resistance against nonadapted powdery mildew fungi and is targeted to papillae deposited at sites of interaction with the fungus. Timely recruitment of PEN3 and other components of penetration resistance to the host-pathogen interface is important for successful defense against this biotrophic pathogen. A forward genetic screen was previously carried out to identify Arabidopsis mutants that mistarget the PEN3 transporter or fail to accumulate PEN3 at sites of attempted powdery mildew penetration. This study focuses on PEN3 mistargeting in the aberrant localization of PEN3 4 (alp4) mutant and identification of the causal gene. In the alp4 mutant, PEN3 accumulates within the endomembrane system in an apparently abnormal endoplasmic reticulum and is not exported into papillae at powdery mildew penetration sites. This targeting defect compromises defenses at the host-pathogen interface, resulting in increased penetration success by a nonadapted powdery mildew. Genetic mapping identified alp4 as an allele of GOLGI DEFECTS 36 (GOLD36), a gene encoding a GDSL-lipase/esterase family protein that is involved in maintaining normal morphology and organization of multiple endomembrane compartments. Genetic complementation confirmed that mutation in GOLD36 is responsible for the PEN3 targeting and powdery mildew penetration resistance defects in alp4. These results reinforce the importance of endomembrane trafficking in resistance to haustorium-forming phytopathogens such as powdery mildew fungi.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Multiple Species of Asteraceae Plants Are Susceptible to Root Infection by the Necrotrophic Fungal Pathogen Sclerotinia sclerotiorum.

The necrotrophic fungal pathogen Sclerotinia sclerotiorum can cause disease on numerous plant species, including many important crops. Most S. sclerotiorum-incited diseases of crop plants are initiated by airborne ascospores produced when fungal sclerotia germinate to form spore-bearing apothecia. However, basal stalk rot of sunflower occurs when S. sclerotiorum sclerotia germinate to form mycelia within the soil, which subsequently invade sunflower roots. To determine whether other plant species in the Asteraceae family are susceptible to root infection by S. sclerotiorum, cultivated sunflower (Helianthus annuus L.) and seven other Asteraceae species were evaluated for S. sclerotiorum root infection by inoculation with either sclerotia or mycelial inoculum. Additionally, root susceptibility of sunflower was compared with that of dry edible bean and canola, two plant species susceptible to S. sclerotiorum but not known to display root-initiated infections. Results indicated that multiple Asteraceae family plants are susceptible to S. sclerotiorum root infection after inoculation with either sclerotia or mycelium. These observations expand the range of plant hosts susceptible to S. sclerotiorum root infection, elucidate differences in root inoculation methodology, and emphasize the importance of soilborne infection to Asteraceae crop and weed species.

A Greenhouse Method to Evaluate Sunflower Quantitative Resistance to Basal Stalk Rot Caused by Sclerotinia sclerotiorum.

Resistance of sunflower to basal stalk rot (BSR) caused by the fungus Sclerotinia sclerotiorum is quantitative, controlled by multiple genes contributing small effects. Consequently, artificial inoculation procedures allowing sufficient throughput and resolution of resistance are needed to identify highly resistant sunflower germplasm resources and to map loci contributing to resistance. The objective of this study was to develop a greenhouse-based method for evaluating sunflower quantitative resistance to BSR that would be simple, space- and time-efficient, high throughput, high resolution, and correlated with field observations. Experiments were conducted with 5-week-old sunflower plants and Sclerotinia-infested millet seed as inoculum to assess the impact of pot size and temperature and to determine the most favorable inoculum rate and placement. Subsequently, an additional experiment was performed to assess the correlation of the greenhouse inoculation procedure with field results by using a panel of 32 sunflower genotypes with known field response to BSR previously determined in multiyear, multilocation artificially inoculated trials. Experimental observations indicated that the newly developed greenhouse inoculation procedure provided improved resolution to identify highly resistant genotypes and was strongly correlated with field observations. This method will be useful for screening of sunflower experimental and breeding materials, disease phenotyping of genetic mapping populations, and evaluation of resistance to different pathogen isolates.

Determination of Virulence Phenotypes of Plasmopara halstedii in the United States.

Downy mildew, caused by Plasmopara halstedii (Farl.) Berl. and de Toni, is an economically important disease in cultivated sunflowers, Helianthus annuus L. Resistance genes incorporated into commercial hybrids are used as an effective disease management tool, but the duration of effectiveness is limited as virulence evolves in the pathogen population. A comprehensive assessment of pathogen virulence was conducted in 2014 and 2015 in the U.S. Great Plains states of North Dakota and South Dakota, where approximately 75% of the U.S. sunflower is produced annually. The virulence phenotypes (and races) of 185 isolates were determined using the U.S. standard set of nine differentials. Additionally, the virulence phenotypes of 61 to 185 isolates were determined on 13 additional lines that have been used to evaluate pathogen virulence in North America and/or internationally. Although widespread virulence was identified on several genes, new virulence was identified on the Pl8 resistance gene, and no virulence was observed on the PlArg, Pl15, Pl17 and Pl18 genes. Results of this study suggest that three additional lines should be used as differentials and agree with previous studies that six lines proposed as differentials should be used in two internationally accepted differential sets. For effective disease management using genetic resistance, it is critical that virulence data be relevant and timely. This is best accomplished when pathogen virulence is determined frequently and by using genetic lines containing resistance genes actively incorporated into commercial cultivars.

Genetic Dissection of Phomopsis Stem Canker Resistance in Cultivated Sunflower Using High Density SNP Linkage Map.

Phomopsis stem canker (PSC) caused by Diaporthe helianthi is increasingly becoming a global threat for sunflower production. In this study, the genetic basis of PSC resistance was investigated in a recombinant inbred line (RIL) population developed from a cross between HA 89 (susceptible) and HA-R3 (resistant). The RIL population was evaluated for PSC disease incidence (DI) in seven screening trials at multiple locations during 2016-2018. The distribution of PSC DI in the RIL population was continuous, confirming a polygenic inheritance of the trait. A moderately high broad-sense heritability (H2, 0.76) was estimated for the trait across environments. In the combined analysis, both the genotype and the genotype × environment interactions were highly significant. A linkage map spanning 1505.33 cM was constructed using genotyping-by-sequencing derived markers. Marker-trait association analysis identified a total of 15 quantitative trait loci (QTL) associated with PSC resistance on 11 sunflower chromosomes, each explaining between 5.24 and 17.39% of the phenotypic variation. PSC resistance QTL were detected in two genomic regions each on chromosomes 3, 5, 13, and 17, while one QTL each was detected in the remaining seven chromosomes. Tightly linked single nucleotide polymorphism (SNP) markers flanking the PSC resistance QTL will facilitate marker-assisted selection in PSC resistance sunflower breeding.

Unraveling the Sclerotinia Basal Stalk Rot Resistance Derived From Wild Helianthus argophyllus Using a High-Density Single Nucleotide Polymorphism Linkage Map.

Basal stalk rot (BSR), caused by the fungus Sclerotinia sclerotiorum, is a serious disease of sunflower (Helianthus annuus L.) in the humid temperate growing areas of the world. BSR resistance is quantitative and conditioned by multiple genes. Our objective was to dissect the BSR resistance introduced from the wild annual species Helianthus argophyllus using a quantitative trait loci (QTL) mapping approach. An advanced backcross population (AB-QTL) with 134 lines derived from the cross of HA 89 with a H. argophyllus Torr. and Gray accession, PI 494573, was evaluated for BSR resistance in three field and one greenhouse growing seasons of 2017-2019. Highly significant genetic variations (p < 0.001) were observed for BSR disease incidence (DI) in all field screening tests and disease rating and area under the disease progress curve in the greenhouse. The AB-QTL population and its parental lines were genotyped using the genotyping-by-sequencing method. A genetic linkage map spanning 2,045.14 cM was constructed using 3,110 SNP markers mapped on 17 sunflower chromosomes. A total of 21 QTL associated with BSR resistance were detected on 11 chromosomes, each explaining a phenotypic variation ranging from 4.5 to 22.6%. Of the 21 QTL, eight were detected for BSR DI measured in the field, seven were detected for traits measured in the greenhouse, and six were detected from both field and greenhouse tests. Thirteen of the 21 QTL had favorable alleles from the H. argophyllus parent conferring increased BSR resistance.

Molecular dissection of resistance gene cluster and candidate gene identification of Pl17 and Pl19 in sunflower by whole-genome resequencing.

Sunflower (Helianthus annuus L.) production is challenged by different biotic and abiotic stresses, among which downy mildew (DM) is a severe biotic stress that is detrimental to sunflower yield and quality in many sunflower-growing regions worldwide. Resistance against its infestation in sunflower is commonly regulated by single dominant genes. Pl17 and Pl19 are two broad-spectrum DM resistance genes that have been previously mapped to a gene cluster spanning a 3.2 Mb region at the upper end of sunflower chromosome 4. Using a whole-genome resequencing approach combined with a reference sequence-based chromosome walking strategy and high-density mapping populations, we narrowed down Pl17 to a 15-kb region flanked by SNP markers C4_5711524 and SPB0001. A prospective candidate gene HanXRQChr04g0095641 for Pl17 was identified, encoding a typical TNL resistance gene protein. Pl19 was delimited to a 35-kb region and was approximately 1 Mb away from Pl17, flanked by SNP markers C4_6676629 and C4_6711381. The only gene present within the delineated Pl19 locus in the reference genome, HanXRQChr04g0095951, was predicted to encode an RNA methyltransferase family protein. Six and eight SNP markers diagnostic for Pl17 and Pl19, respectively, were identified upon evaluation of 96 diverse sunflower lines, providing a very useful tool for marker-assisted selection in sunflower breeding programs.

PMR5, an acetylation protein at the intersection of pectin biosynthesis and defense against fungal pathogens.

Powdery mildew (Golovinomyces cichoracearum), one of the most prolific obligate biotrophic fungal pathogens worldwide, infects its host by penetrating the plant cell wall without activating the plant's innate immune system. The Arabidopsis mutant powdery mildew resistant 5 (pmr5) carries a mutation in a putative pectin acetyltransferase gene that confers enhanced resistance to powdery mildew. Here, we show that heterologously expressed PMR5 protein transfers acetyl groups from [14 C]-acetyl-CoA to oligogalacturonides. Through site-directed mutagenesis, we show that three amino acids within a highly conserved esterase domain in putative PMR5 orthologs are necessary for PMR5 function. A suppressor screen of mutagenized pmr5 seed selecting for increased powdery mildew susceptibility identified two previously characterized genes affecting the acetylation of plant cell wall polysaccharides, RWA2 and TBR. The rwa2 and tbr mutants also suppress powdery mildew disease resistance in pmr6, a mutant defective in a putative pectate lyase gene. Cell wall analysis of pmr5 and pmr6, and their rwa2 and tbr suppressor mutants, demonstrates minor shifts in cellulose and pectin composition. In direct contrast to their increased powdery mildew resistance, both pmr5 and pmr6 plants are highly susceptibile to multiple strains of the generalist necrotroph Botrytis cinerea, and have decreased camalexin production upon infection with B. cinerea. These results illustrate that cell wall composition is intimately connected to fungal disease resistance and outline a potential route for engineering powdery mildew resistance into susceptible crop species.

Introgression and monitoring of wild Helianthus praecox alien segments associated with Sclerotinia basal stalk rot resistance in sunflower using genotyping-by-sequencing.

Sclerotinia basal stalk rot (BSR) and downy mildew are major diseases of sunflowers worldwide. Breeding for BSR resistance traditionally relies upon cultivated sunflower germplasm that has only partial resistance thus lacking an effective resistance against the pathogen. In this study, we report the transfer of BSR resistance from sunflower wild species, Helianthus praecox, into cultivated sunflower and molecular assessment of the introgressed segments potentially associated with BSR resistance using the genotyping-by-sequencing (GBS) approach. Eight highly BSR-resistant H. praecox introgression lines (ILs), H.pra 1 to H.pra 8, were developed. The mean BSR disease incidence (DI) for H.pra 1 to H.pra 8 across environments for four years ranged from 1.2 to 11.1%, while DI of Cargill 270 (susceptible check), HA 89 (recurrent parent), HA 441 and Croplan 305 (resistant checks) was 36.1, 31.0, 19.5, and 11.6%, respectively. Molecular assessment using GBS detected the presence of H. praecox chromosome segments in chromosomes 1, 8, 10, 11, and 14 of the ILs. Both shared and unique polymorphic SNP loci were detected throughout the entire genomes of the ILs, suggesting the successful transfer of common and novel introgression regions that are potentially associated with BSR resistance. Downy mildew (DM) disease screening and molecular tests revealed that a DM resistance gene, Pl17, derived from one of the inbred parent HA 458 was present in four ILs. Introgression germplasms possessing resistance to both Sclerotinia BSR and DM will extend the useful diversity of the primary gene pool in the fight against two destructive sunflower diseases.

Follicular lymphoma presenting as scalp mass deformity: Case Report and Review of the literature.

Lymphoma presenting as a scalp mass is a rare but serious medical condition mandating aggressive treatment and neurosurgical intervention. We report a case of 53-year-old male who presented with a large right sided frontal scalp mass and a smaller mass located on the left frontal scalp. After discussion with the patient, it was decided to resect the larger mass for definitive diagnosis. After subtotal resection of the mass, biopsy revealed WHO grade 1 follicular lymphoma (FL), diffuse pattern stage IV. The patient was subsequently treated with 4 grays (Gy) of palliative radiotherapy over 2 fractions to the right frontal scalp and systemic chemo-immunotherapy (6 cycles) followed by rituximab maintenance. Lumbar puncture to obtain cerebrospinal fluid was done a month after therapy began and the results were negative for spread of malignant cells. Approximately 3 months after initiation of therapy, PET/CT showed no evidence of active malignancy and MRI revealed a complete internal resolution of the enlarged right frontal scalp mass. We use this case to provide a detailed discussion regarding disease pathophysiology, early diagnosis, and management.

Phosphorylation is required for the pathogen defense function of the Arabidopsis PEN3 ABC transporter.

The Arabidopsis PEN3 ABC transporter accumulates at sites of pathogen detection, where it is involved in defense against a number of pathogens. Perception of PAMPs by pattern recognition receptors initiates recruitment of PEN3 and also leads to PEN3 phosphorylation at multiple amino acid residues. Whether PAMP-induced phosphorylation of PEN3 is important for its defense function or focal recruitment has not been addressed. In this study, we evaluated the role of PEN3 phosphorylation in modulating the localization and defense function of the transporter. We report that PEN3 phosphorylation is critical for its function in defense, but dispensable for recruitment to powdery mildew penetration sites. These results indicate that PAMP-induced phosphorylation is likely to regulate the transport activity of PEN3.

An Arabidopsis Lipid Flippase Is Required for Timely Recruitment of Defenses to the Host-Pathogen Interface at the Plant Cell Surface.

Deposition of cell wall-reinforcing papillae is an integral component of the plant immune response. The Arabidopsis PENETRATION 3 (PEN3) ATP binding cassette (ABC) transporter plays a role in defense against numerous pathogens and is recruited to sites of pathogen detection where it accumulates within papillae. However, the trafficking pathways and regulatory mechanisms contributing to recruitment of PEN3 and other defenses to the host-pathogen interface are poorly understood. Here, we report a confocal microscopy-based screen to identify mutants with altered localization of PEN3-GFP after inoculation with powdery mildew fungi. We identified a mutant, aberrant localization of PEN3 3 (alp3), displaying accumulation of the normally plasma membrane (PM)-localized PEN3-GFP in endomembrane compartments. The mutant was found to be disrupted in the P4-ATPase AMINOPHOSPHOLIPID ATPASE 3 (ALA3), a lipid flippase that plays a critical role in vesicle formation. We provide evidence that PEN3 undergoes continuous endocytic cycling from the PM to the trans-Golgi network (TGN). In alp3, PEN3 accumulates in the TGN, causing delays in recruitment to the host-pathogen interface. Our results indicate that PEN3 and other defense proteins continuously cycle through the TGN and that timely exit of these proteins from the TGN is critical for effective pre-invasive immune responses against powdery mildews.

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing.

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 µg DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., > 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.