Alex Zaccaronhttps://doi.org/10.1094/ISMPMI-2023-1Cladosporium fulvum causes tomato leaf mold and has been extensively used in the past as a model species to study plant-microbe interactions. Although the first chromosome-scale reference genome of the fungus was released in 2022, still little is known about how its genome architecture and structural variations (SVs) thereof impact its virulence. In this study, we used PacBio HiFi to sequence the genomes of four additional C. fulvum isolates and further assembled them at chromosome level. Comparative genome analyses revealed high chromosomal synteny among the five isolates, and a set of 13 core and two dispensable chromosomes, one of which carries pseudogenized copies of effector genes and likely emerged by duplication of subtelomeric regions of core chromosome. Between 14906 and 14993 genes were predicted in each C. fulvum genome with an estimated completeness of >99%. A pangenome analysis of the five isolates revealed a low number of 331 accessory genes, indicating high conservation of gene content among isolates of the fungus. An analysis of SVs showed no enriched of effectors or of other pathogenicity-related genes in these regions. However, SVs in subtelomeric regions affected virulence by prompting the loss of effector genes residing in them, as we have found is the case for the Avr9 effector gene of C. fulvum. Collectively, these results provide new insights of how genomic SVs can contribute to virulence of fungal pathogens. View Poster
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Alex Zaccaronhttps://doi.org/10.1094/ISMPMI-2023-2Erysiphe necator causes grape powdery mildew, globally the most important fungal disease on grapevines. The current reference genome assembly of the pathogen is highly fragmented due to its repetitive DNA content, which prevents a detailed analysis of its genomic architecture. Here, we present an 81.1 Mb genome assembly for E. necator that is 98% complete and consists of 34 scaffolds, 11 of which represent complete chromosomes. Analysis of the chromosomes’ content showed that they all contain large centromeric-like regions that account for 15.8% of the genome. Repeats and transposable elements (TEs) represent 62.7% of their content and, while they are almost evenly interspersed outside centromeric and telomeric regions, they massively overlap with regions of annotated genes, indicating that they could have a significant impact on their evolution. Indeed, abundant gene duplicates were observed, particularly in genes encoding candidate secreted effector proteins (CSEPs) that could have been spawned by TE activity. Copies of duplicated CSEP-encoding genes were further found to be frequently clustered in the genome, suggesting recurrent events of local gene duplications and adaptive variation. Finally, in accordance with the obligate biotrophic nature of E. necator,abundant losses in genes needed to synthesize metabolites that can be obtained directly through the host environment were also observed. These and other genomic architectural features of E. necator will be discussed. View Poster
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Andrea Carobbihttps://doi.org/10.1094/ISMPMI-2023-3Pantoea agglomerans pv. betae (Pab) is a bacterial pathogen causing development of galls on roots of beet and gypsophyla plants. The Pab genome contains a gene cluster predicted to encode a complete type VI secretion system (T6SS) that delivers toxic effectors into neighboring cells. By secretion and competition assays, we provide evidence that the Pab T6SS is a functional antibacterial system that enables Pab to outcompete other plant-associated bacteria. Antibacterial effectors and immunity proteins are encoded inside three genomic islands that also contain arrays of orphan immunity genes or toxin and immunity pairs. We have discovered a specialized effector that contains a C-terminal catalytically active glucosaminidase domain, which degrades prey peptidoglycan. We also demonstrated that a novel antibacterial T6SS effector and immunity pair are encoded by an operon located at the end of the Pab T6SS cluster. Two putative effectors were identified outside the main cluster and share a similar N-terminal domain which we found to be conserved also in many Enterobacteria. This domain is essential for T6SS1-dependent secretion of certain effectors shown to be toxic when expressed in sister bacteria lacking the appropriate effector and immunity genes. Our observations indicate that the Pab T6SS1 operates as an antibacterial system that employs a lysozyme-like effector and other effectors, encoded both within and outside the T6SS1 cluster, to eliminate rival bacteria. View Poster
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Andrew Benthttps://doi.org/10.1094/ISMPMI-2023-4RSoybean cyst nematode (SCN) is the most yield-reducing pathogen of soybean. Resistance based on the complex rhg1-b haplotype has been the primary control measure, but gradual SCN evolution is incrementally eroding rhg1-b efficacy. rhg1-b carries ten copies of a ~31 kb chromosomal segment with three different genes that contribute to resistance. We are functionally dissecting the rhg1-b α-SNAP to understand and possibly improve this novel defense mechanism. α-SNAP is a housekeeping protein with C-terminal amino acids that are conserved across multicellular eukaryotes. a-SNAP interacts with NSF to recycle SNARE protein bundles that mediate vesicle fusion to target membranes. The rhg1-b a-SNAPRhg1HC protein differs from canonical α-SNAPs at its C-terminus and apparently poisons the nematode-plant biotrophy when its level increases 10-20 fold at SCN feeding sites (syncytia). We will describe our site-directed mutagenesis to alter and test functional C-terminal residues and potentially block SCN effector action against rhg1-b α-SNAP. We are also investigating the mechanisms by which a-SNAPRhg1HC abundance is elevated at syncytia, and will report an in situ assay system to obtain spatially resolved transcript abundance data at syncytia via confocal microscopy. This system is now being used to dissect rhg1-b promoter elements as one logical first step to understanding mechanisms of infection-site upregulation and potentially allow promoter modification to enhance SCN resistance. View Poster
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Chuntao Yinhttps://doi.org/10.1094/ISMPMI-2023-5RRhizosphere microbiota, referred to as the second genome of plants, are crucial to plant health. Increasing evidence reveals that plants can change their rhizosphere microbiome and promote microbial activity to reduce plant disease. However, how plant and phytopathogens factor in combination to structure the rhizosphere microbiome and govern microbial selection for adaptation to disease stress remains incompletely understood. In this study, rhizosphere microbiota from successive wheat plantings under the pressure of the soilborne pathogen Rhizoctonia solani AG8 were characterized. Amplicon sequence analyses revealed that bacterial and fungal communities clustered by planting cycles. The addition of AG8 enhanced the separation of the rhizosphere microbiota. The alpha diversity of bacteria and fungi significantly decreased over planting cycles. Compared with rhizosphere bacterial communities, AG8 was a major driver structuring fungal communities. Pathogen-infected monocultures enriched a group of bacterial genera with potential antagonistic activities or abilities for plant growth promotion or nitrogen fixation. Further, eleven bacterial species exhibited antagonistic activities toward Rhizoctonia spp., and four of them displayed broad antagonism against multiple soilborne fungal pathogens. These findings support the potential to improve plant health through manipulating rhizosphere microbiota. View Poster
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Cintia Sagawahttps://doi.org/10.1094/ISMPMI-2023-6Citrus greening or Huanglongbing (HLB) disease is caused by the bacteria Candidatus Liberibacter spp. which is a major threat to the citrus industry worldwide. This has led to the generation of a large number of genomic databases characterizing HLB at the molecular level. Based on the genomic information of 71 published transcriptomic and proteomic datasets we selected 1,200 potential susceptibility genes associated with HLB in citrus. By using CRISPR/Cas9 technology combined with a multiplex approach, we generated 300 constructs targeting a combination of four of those genes in each construct. We currently have a population of over 3,000 transformed lines of Carrizo, a commercial rootstock hybrid (Citrus sinensis 'Washington' sweet orange X Poncirus trifoliata) and population of Valencia sweet orange lines with a diversified combination of mutations. To aid in genotyping, our group is performing a whole genome sequencing of the hybrid Carrizo. Biallelic/homozygous mutants were confirmed by Sanger sequencing of target sites and alignment with respective genomes. Confirmed mutants are currently being tested for bacterial resistance. Identification and stacking of susceptibility gene mutations will be valuable in developing tolerance to HLB and these mutations can subsequently be introduced into other economically significant citrus cultivars and evaluated in the field. View Poster
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Daniel Monino Lopezhttps://doi.org/10.1094/ISMPMI-2023-7Phytophthora infestans is a devastating pathogenic oomycete that causes potato late blight. Wild Solanum species are known to be a major source of resistance against P. infestans. In this study, we described a late blight broad spectrum resistant Solanum agrimoniifolium accession, resistant to 26 out of 27 tested P. infestans isolates. Here, we cloned the resistance gene located on the long arm of chromosome 7. Unlike most Rpi genes, the new resistance gene does not code for an NLR but for Lyst-Interacting Protein 5 (LIP5), which is an essential component of the endosomal sorting complex required for transport (ESCRT) pathway involved in the formation of multivesicular bodies (MVBs). Allele mining of LIP5 in other S. agrimoniifolium accessions identified only two amino acid polymorphisms between the proteins encoded by the resistant SaLIP5-1 and susceptible SaLIP5-2 alleles. We hypothesized that SaLIP5-1 is an effector target guarded by an NLR. Indeed, we found three different P. infestans effectors that interact with SaLIP5 in planta. Furthermore, we demonstrated that SaLIP5-1-mediated resistance requires helper NLRs (NRCs), suggesting that SaLIP5-1 is indeed guarded by an NLR. This study provides unique insights into how late blight resistance evolves among crop wild relatives and is essential for the introgression of the S. agrimoniifolium resistance into the S. tuberosum background. View Poster
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Eilyn Menahttps://doi.org/10.1094/ISMPMI-2023-8Soybean is an important crop worldwide, its production is limited by soybean stem canker disease caused by Diaporthe caulivora. In this study, two contrasting soybean cultivars, Williams (susceptible) and Génesis 5601 (resistant) were compared in response to infection with D.caulivora. Génesis 5601 was more resistant to fungal infection than Williams, evidenced by reduced length lesions, disease severity index and pathogen biomass. Transcriptional profiling was performed in D.caulivora-inoculated and control tissues. In total, 2322 and 1855 genes were differentially expressed in Génesis 5601 and Williams, respectively. Under basal conditions Génesis 5601 presents higher expression of genes related to plant defense. At 8 hours post inoculation an earlier defense response was activated in Génesis 5601, demonstrated by upregulation of 1028 compared to 434 genes in Williams. Resistance to D.caulivora was associated with perception of the pathogen by surface and intracellular receptors, and defense activation through transcription factors, biosynthesis of phenylpropanoids, hormones, small heat shock proteins and genes with different roles in defense. Moreover, cell wall modifications, accumulation of phenolic compounds and reactive oxygen species were observed in stems of inoculated plants compared to the control. These findings provide novel insights into soybean molecular defense developed to control this pathogen, and a foundation for improving resistance in breeding programs. View Poster
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Eleni Koseoglouhttps://doi.org/10.1094/ISMPMI-2023-9Tomato bacterial canker caused by Clavibacter michiganensis (Cm) is considered to be one of the most destructive bacterial diseases of tomato. To date, no resistance to the pathogen has been identified. While several molecular studies have identified (Cm) bacterial factors involved in disease development, the plant genes and mechanisms associated with susceptibility of tomato to the bacterium remain largely unknown. Here, we show for the first time that tomato gene SlWAT1 is a susceptibility gene to Cm. We inactivated the gene SlWAT1 through RNAi and CRISPR/Cas9 to study changes in tomato susceptibility to Cm. Furtermore, we analysed the role of the gene in the molecular interaction with the pathogen. Our findings demonstrate that SlWAT1 functions as an S gene to genetically diverse Cm strains. Inactivation of SlWAT1 reduced free auxin contents and ethylene synthesis in tomato stems and suppressed the expression of specific bacterial virulence factors. However, CRISPR/Cas9 slwat1 mutants exhibited severe growth defects. The observed reduced susceptibility is possibly a result of downregulation of bacterial virulence factors and reduced auxin contents in transgenic plants. This is the first time it has been shown that an S gene can regulate the expression of virulence factors. View Poster
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Filippo Bincihttps://doi.org/10.1094/ISMPMI-2023-10The recognition of different microbe-associated molecular patterns triggers in the plant the activation of either an immune response or an accommodation program. In both types of responses, Ca2+ is a crucial intracellular messenger, mediating the early stages of the respective signalling pathways. In this work, we analyzed the cytosolic and nuclear Ca2+ changes activated by a set of chitin-related oligomers in different genetic backgrounds of Lotus japonicus roots by using specifically targeted aequorin-based Ca2+ reporters. By means of pharmacological and genetic approaches, we dissected the plant Ca2+ responses into two separable and temporally distinct components. A complementary approach carried out via a cameleon-based bioassay in Medicago truncatula root organ cultures further supports the occurrence of different temporal phases in the observed Ca2+ transients. While the second phase of the Ca2+ change represents the well-known Ca2+ spiking phenomenon, we show that the occurrence of the rapid first phase, which critically depends on the elicitor concentration, correlates with the activation of plant immunity marker genes. Our data provide clues to a better understanding of the subtle boundaries between symbiotic and immunity responses in plant-fungus interactions. View Poster
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Francisca Blanco-Herrerahttps://doi.org/10.1094/ISMPMI-2023-11Aphids are phloem-feeding insects that represent a major threat to world agriculture. The feeding strategy involves a slender stylet that probes the host tissues using intercellular spaces as the main pathways to reach the phloem. It is thought that pectin degradation facilitates stylet penetration through the cell walls. However, host plants may use this feeding mechanism since they have evolved to sense the byproducts of pectin degradation called oligogalacturonides (OGs) and trigger a defense response. OGs are well-known damage-associated molecular patterns (DAMPS) that enhance plant resistance. However, whether plants use this defense mechanism against aphids is unknown yet. We show that pectin and pectin-modifying enzymes were significantly altered during the plant-aphid interaction. As a result, M. persicae infestation induced a significant increase in total PME and PL activities, and a decrease in the methyl-esterification degree of pectin. In addition, OGs treatments in Arabidopsis increase the resistance to M. persicae infestation by reducing their offspring number, settling preference, and feeding performance. This enhanced resistance was related to a high accumulation of ROS and callose deposits, an increase in the transcript level related to salicylic acid (SA) pathway genes, and the activation of the SA signaling pathway. View Poster
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Giovanni Mianhttps://doi.org/10.1094/ISMPMI-2023-12Kiwifruit Vine Decline Syndrome (KVDS) is the most important soil-borne disease within Italian kiwifruit industry, causing €300,000 of economic losses in 2020 alone. So far, the KVDS’ aetiological agents belong primarily to the oomycetes. Since no effective management strategies exist yet, the most promising approach to overcoming KVDS is therefore the use of resistant species as rootstocks or for breeding programmes. Several Actinidia genotypes showing different resistance’ rates to KVDS were grown in disease-promoting soils. A metabarcoding approach was set up to identify the oomycetes associated with KVDS and investigate whether the primary involved specie(s) can vary according to plant genotype. Our results clearly showed significant differences between genotypes in terms of oomycetes detected in both plant rhizosphere and endosphere, which correlated strongly with the symptoms displayed. We found that the resistance of A. macrosperma to KVDS is related to its ability to shape the pathobiome, particularly as far as the endosphere is concerned. Under our conditions, Phytophthora sp. subclade7b (Yang et al., 2017) was found predominantly in sensitive genotypes; whilst Globisporangium intermedium was detected mainly in asymptomatic plants, suggesting that the latter might compete with Phytophthora sp. recruitment in resistant plants, and explain the occurrence of symptoms and resistance. View Poster
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Heike Sederoffhttps://doi.org/10.1094/ISMPMI-2023-13Arbuscular mycorrhizal symbiosis (AM) is a beneficial trait originating with the first land plants. The ability to host AM has since been lost from diverse plant species. Genes in the Common Symbiosis Pathway that are essential to establish AM hosting were lost from Brassicaceae along with the trait itself, including Interacting Protein of DMI3 (IPD3), a key transcription factor connecting upstream signaling of AM fungal presence to the downstream gene-regulatory network for AM functions. We generated transgenic Arabidopsis plants expressing the DNA-binding domain of IPD3 and used phenotypic and transcriptome analysis to characterize the effect of IPD3 expression in Arabidopsis in the presence and absence of AM fungus Rhizophagus irregularis. We compared these results to the AM-host model Lotus japonicus and its ipd3 knockout mutant cyclops-4. Despite its long history as a non-AM species, restoring IPD3 expression to Arabidopsis significantly altered transcription related to growth and defense, and delayed flowering time. Our comparative transcriptomic results indicate that some genetic connections for IPD3 remain conserved in Arabidopsis despite the long evolutionary time since its loss. View Poster
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Hsuan Paihttps://doi.org/10.1094/ISMPMI-2023-14Nucleotide-binding domain leucine-rich repeat (NLR) proteins play a vital role in plant and metazoan innate immunity, oligomerizing into multiprotein complexes known as resistosomes or inflammasomes upon activation. NLRs can function as individual singletons or in higher-order configurations, such as pairs, or networks, which exhibit a sensor-helper dynamic. While recent structural studies have provided ground-breaking insights into the molecular mechanisms of singleton NLR activation, the biochemical mechanisms underlying the activation of paired and networked NLRs remain unclear. In this study, we leveraged blue native polyacrylamide gel electrophoresis (BN-PAGE) to investigate NLR activation, focusing on the Pik1/Pik2 system and the NRC (NLR required for cell death) network as models for paired and networked NLRs, respectively. We found that the engineered Pikm-1/Pikm-2 sensor-helper pair (aka Pikobodies) constitutively form an oligomeric hetero-complex, which incorporates its cognate effector for activation. In contrast, the sensor/helper pairs in the NRC network follow an activation-and-release model, in which sensor NLRs mediate the formation of helper NRC homo-oligomers upon effector perception. We will introduce the method pipeline for establishing BN-PAGE assays for studying NLR biology and discuss our results, which highlight BN-PAGE as a valuable and versatile tool for visualizing NLR activation and studying oligomeric resistosomes and their components. View Poster
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Jahed Ahmedhttps://doi.org/10.1094/ISMPMI-2023-15As sessile organisms, plants always have to deal with different environmental stimuli, the generation of reactive oxygen species (ROS) and calcium (Ca2+) flux as second messengers are one of the common strategies to response against such stimuli. Upon perception of pathogen, molecular warning signals propagate inside the cell and throughout the plant tissue to trigger defense mechanisms. However, the role of ROS and Ca2+ during viral infection process and associated signaling mechanism remains largely unknown. Viruses are obligate intracellular pathogens that hijack host machineries to facilitate their replication and propagation across the plant through plasmodesmata (PD), communication channels bridging the plant cells. Here, we explored the mutual role of generated ROS and Ca2+ signals using biosensors and study the crosstalk between the two signals during viral infection. The results suggest that virus induces the ROS and Ca2+ elevation in virus infected and distal area on plant leaves. Later virus hijacks the host ROS/Ca2+ machineries to facilitate their propagation throughout plant by opening PD. Using genetics, genome editing technology and super-resolution microscopy techniques, we showed that virus perturbs the plasma membrane (PM) organization by interrupting PM localized protein nanodomains that contribute to the ROS and Ca2+ signaling as plant defense response. View Poster
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Jake Schumacherhttps://doi.org/10.1094/ISMPMI-2023-16Improved incorporation of biological nitrogen fixation into agricultural systems via the development of the legume-rhizobia symbiosis would greatly aid efforts to minimize pollution from the overuse of nitrogen fertilizers. Highly competitive rhizobia fix nitrogen for legumes within symbiotically formed root nodules. Rhizobia must be both effective and competitive to benefit its intended host. However, rhizobia that are the most effective at fixing nitrogen are not necessarily the most competitive for nodule formation. This study investigates the genomic basis of competitiveness and effectiveness in the model rhizobia Sinorhizobium meliloti which forms a symbiotic relationship with both alfalfa and the model legume Medicago truncatula. S. meliloti has a tripartite genome consisting of its main chromosome, a pSymA megaplasmid, and a pSymB chromid. Hybrid strains of S. meliloti were created by transferring pSymA, pSymB, and both pSymA and pSymB from a defined set of strains into a wildtype background. By evaluating the transfer of competitiveness and effectiveness phenotypes from natural strains to the wildtype background, we are able to gain insight into the genomic basis of these traits. Our data lead us to propose a model wherein the symbiotic megaplasmid pSymA is the primary reservoir for genetic determinants of effectiveness, while competitiveness genes are primarily found on the pSymB chromid and the main chromosome. View Poster
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Jennette Codjoehttps://doi.org/10.1094/ISMPMI-2023-17Pennycress (Thlaspi arvense) is a weed that is being domesticated into a winter cover crop for use in corn-soy rotations in the Midwestern U.S. Pennycress seeds can be sold as an oilseed for biofuel and animal feed applications, making it an attractive ‘cash cover crop’. Like other Brassica species, pennycress is susceptible to infection by the necrotrophic fungus Sclerotinia sclerotiorum, the causal agent of white mold or Sclerotinia stem rot disease. We are investigating the use of small antifungal peptides to control S. sclerotiorum infection in pennycress. One endogenous peptide, plant defensin 2.2 (TaPDF2.2), inhibits the in vitro growth of S. sclerotiorum field isolate at a minimum inhibitory concentration (MIC) of 24µM. Another, nodule cysteine-rich peptide 13 from chickpea (CaNCR13), is even more effective, exhibiting in vitro antifungal activity with an MIC of 3µM or 24µM, depending on the disulfide connectivity in the peptide. We have generated transgenic 35S:TaPDF2.2 and 35S:CaNCR13 pennycress lines, and I will present whether overexpression of these antifungal peptides is able to improve the resistance of pennycress to S. sclerotiorum. View Poster
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Jiuyue Panhttps://doi.org/10.1094/ISMPMI-2023-18Plants represent the nutritional basis of virtually all life on earth. Protein-rich foods from crop plants are a global megatrend essential for sustaining an increasing human population and counteracting climate change. While the genomes of crops are increasingly elucidated, little is known about crop proteomes – the entirety of proteins that execute and control nearly every aspect of life. Therefore, we have launched the visionary international doctoral program “LRTN360 - The Proteomes that Feed the World”, which was conceived at TUM and is now funded by the Elite Network of Bavaria. One of the program’s overarching aims is to map the proteomes of all major tissues and organs of the 100 crop plants most important for human nutrition, thereby creating a Crop Proteome Atlas of extremely high value to academia as well as the agricultural, food and beverage, and pharmaceutical industries. Besides creating this Crop Proteome Atlas as a team effort, all PhD candidates work on individual research projects investigating detailed aspects of plant proteomes. One of these individual projects will be presented in more detail: comparative proteomics and phosphoproteomics analysis of rice, tomato and pea roots colonized by arbuscular mycorrhiza fungi to understand the underlying signal transduction mechanisms. The raw mass spectrometric data, along with the protein identification and quantification results will be made publicly available on PRIDE and in ProteomicsDB on a regular basis. View Poster
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Jogi Madhuprakashhttps://doi.org/10.1094/ISMPMI-2023-19Plant nucleotide-binding leucine-rich repeat (NLR) proteins are critical components of the plant immune system that recognize and respond to pathogen attacks. Upon immune activation, NLRs form higher order complexes termed resistosomes. Parasites can counteract host immunity by suppressing helper NLR proteins that function as central nodes in immune receptor networks. We previously reported that the Phytophthora infestans effector AVRcap1b can suppress cell death mediated cell surface and intracellular immune receptors by targeting helper NLRs NRC2/3. AVRcap1b associates with host NbTOL9a, a protein involved in ESCRT mediated vesicle trafficking, which acts as a negative regulator of NRC signaling. NbTOL9a is genetically required for full AVRcap1b immune suppression. The precise mechanism by which AVRcap1b suppresses NRCs is not fully understood. Here we obtained the crystal structure of AVRcap1b bound to the N-terminal ENTH domain of NbTOL9a. Mutating the AVRcap1b-NbTOL9a binding interface abolishes AVRcap1b immune suppression. We show that while AVRcap1b does not suppress NRC oligomerization, it specifically associates with activated, but not inactive, NRC2. We propose that AVRcap1b bridges the host ESCRT trafficking machinery and activated NRC2 complexes to suppress NRC-mediated cell death. Our results provide insights into how plant parasites can inhibit NLR-mediated signaling resulting in a broad suppression of both cell surface and intracellular immunity. View Poster
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Juarez Tomazhttps://doi.org/10.1094/ISMPMI-2023-20ABA role in plant response to biotic stresses is controversial: induces resistance in some pathosystems and susceptibility in others, but for sedentary nematodes, such as Meloidoyne paranaensis, data are scarce. The aim of this study was to evaluate the effect of ABA on Arabidopsis thaliana defense against M. paranaensis. Col-0 was treated with 80 µM of ABA at 24 hours before and 7 days after inoculation with 180 J2/eggs of M. paranaensis. Specimens were counted 35 days after inoculation, data were submitted to variance analysis (ANOVA) and grouped using Scott-Knott test (p≥0.05). To evaluate the direct effect of ABA on the nematode, 20 specimens (J2) were treated 80µM of ABA during 24 and 48h and the data were submitted to ANOVA and compared by Tukey test (p≥0.05). Mutants for ABA biosynthesis, signaling and receptors were challenged with M. paranaensis and evaluated as described above. Data were submitted to ANOVA and means compared by Student's t test (p≥0.05). Reduction of 58.3% in the nematode population when ABA was applied 24 hours before inoculation corroborated with the nematode increase observed in aba2-1 mutant. ABA did not promote nematostatic or nematicidal effect on the nematode, however, the nematode population increase and decrease in the sextuple mutant and abi4-1, respectively, indicates that the plant's defense responses involve the perception of ABA as signaling molecule. Significant differences in the other evaluated mutants were not observed. View Poster
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Juliana Gonzalez-Tobonhttps://doi.org/10.1094/ISMPMI-2023-21Bacteria belonging to the Dickeya genus cause blackleg and soft rot symptoms on many plant hosts, including potato. During infection the pathogen faces a variety of environmental conditions, and it is unclear which genes allow for its successful growth in them. To identify the genes required for fitness in potato stems, we constructed and evaluated randomly bar-coded transposon mutant (RB-TnSeq) libraries of Dickeya dianthicola and Dickeya dadantii. We identified 43 and 95 genes whose predicted functions impacted the fitness of D. dadantii and D. dianthicola, respectively. Most mutants that were less fit than the wild-type were disrupted in genes related to chemotaxis and motility, suggesting the proteins encoded by these genes are important for survival in potato stems. Additionally, genes involved in processes not previously shown to impact growth in the stem, such as iron sequestration, cellular transport, and transcription, were identified. Also virulence factors, such as srfA-B-C, were shown to have a strong positive impact in growth in the stem. Comparing this RB-TnSeq data set with others generated from multiple in vitro growth conditions and potato tubers allowed us to identify genes required for growth in planta and not in vitro, such as members of the pectin-degradation pathway (kduI and kduD). This data provides important insights into the mechanisms used by Dickeya when interacting with and colonizing plants, and thus might provide targets for management.View Poster
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Juliet Ocholahttps://doi.org/10.1094/ISMPMI-2023-22Iron is a crucial nutrient for plant growth but its bioavailability in soil is low above pH~6. Conversely, too much iron results in toxicity and unrestricted pathogen growth. Plants, bacteria, and fungi produce extracellular secondary metabolites (siderophores) to make iron available for uptake and to compete for this nutrient. Depending on the structures, siderophores can increase or reduce iron bioavailability to plants and affect the plant’s immune system. However, we do not clearly understand which siderophore structures exist in the plant rhizosphere and what their effect is on plant iron availability or immunity. Here we use a library of >200 genome-sequenced bacteria isolated from the roots of Arabidopsis thaliana and wheat varieties to characterize the diversity of siderophores and iron-reducing compounds. Putative siderophore structures were first predicted by analyzing gene clusters with bioinformatics tools. Bacteria were then cultured individually in defined iron-limited media to determine quantities and structures of siderophores and reducing metabolites. High-resolution LC-MS analysis of metabolites in culture supernatants showed that ~66% of bacteria produced siderophores spanning a range of known families but also highly unique structures. We discuss the potential use of these compounds in alleviating plant iron stress in iron-deficient and replete conditions and their application in improving plant defense responses against pathogenic microbes. View Poster
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Léa Monge Waleryszakhttps://doi.org/10.1094/ISMPMI-2023-23Gram-negative bacterial pathogens inject variable repertoires of effectors into host cells to interfere with defense responses. Among those, the Yersinia outer protein J (YopJ) effector family of acetyltransferases, produced by diverse animal and plant bacterial pathogens, promote pathogen virulence by acetylating specific host components. However, the range of host processes that YopJ effectors can interfere with remains elusive. Thus, we developed a proximity-dependent protein labeling involving the Turbo biotin ligase for identifying the interactomes of two well-characterized YopJ members: the Ralstonia pseudosolanacearum PopP2 and its close homolog, XopJ6 from Xanthomonas campestris. Both of them are recognized by the Arabidopsis RRS1/RPS4 pair through manipulation of an integrated WRKY domain that mimics effector primary targets, the WRKY transcription factors. Interestingly, a single residue substitution in a XopJ6 natural variant disrupts physical interaction with WRKY proteins, enabling XopJ6 to avoid host recognition while retaining XopJ6 virulence functions, likely through interference with components other than WRKYs. The different PopP2 and XopJ6 variants fused with Turbo will be expressed in both N. benthamina and Arabidopsis. Alternatively, we developed an approach consisting in Pseudomomas fluorescens-mediated delivery of the Turbo fusion proteins to remain as close as possible to the level of effectors injected in plant cells by pathogenic bacteria. View Poster
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Lingwei Wanhttps://doi.org/10.1094/ISMPMI-2023-24cynT (PSPTO_5255) encodes a carbonic anhydrase whose expression is induced by calcium in Pseudomonas syringae pv. tomato DC3000. Previously we showed cynT contributes to growth of DC3000 and symptom development in tomato plants. To understand the mechanism of how cynT impacts the virulence of DC3000, global transcriptome analysis (RNA-Seq) was performed for the cynT mutant and wild-type (WT). The expression of many virulence-related genes was impacted when cynT was deleted. On rich media (nutrient broth agar) supplemented with calcium, 73 genes were upregulated, and 56 genes were downregulated in the ΔcynT strain compared to the WT. On minimal media (mannitol-glutamate agar) supplemented with calcium, 495 genes were upregulated, and 253 genes were downregulated in the ΔcynT strain compared to the WT. Comparing the datasets, we found that the regulation of 31 genes by cynT was independent of the growth condition, however many of these genes showed opposite patterns of expression, including PSPTO_2870 and PSPTO_2871. PSPTO_2870 and PSPTO_2871 are predicted orthologs of a putative virulence gene, srfA, and srfB respectively in P. syringae pv. syringae B728. Little is known about the functions of these genes in DC3000. Knockout mutants of srfABC operon were constructed in DC3000 and currently being characterized. The results indicate that the carbonic anhydrase, CynT is a key regulator for bacterial factors related to pathogenesis and virulence. View Poster
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Luca Capriottihttps://doi.org/10.1094/ISMPMI-2023-25Fungal and oomycetes pathogens are the major phytopathological problem in grapes cultivation and their control is mainly addressed by performing repeated fungicide treatments. Post-transcriptional gene silencing driven by the mechanism of RNA interference (RNAi) represents a powerful alternative, that can be employed to reduce the virulence of the pathogen. Considering modern viticulture, which is based on the cultivation of grapevine cultivars grafted onto hybrid Vitis rootstocks, the opportunity of having rootstocks capable of producing active long or small dsRNAs targeting fungal RNAs could have important implications as new defense strategies. The generation of grapevine plants expressing hairpin gene constructs aiming at the downregulation of Botrytis cinerea Dicer-like genes 1 and 2 has been optimized in the Thompson Seedless cultivar. The plants composed by a combination of different independent transformed lines used as rootstock down to Wild-type scions have been demonstrated to show a major tolerance to grey mold in detached leaves experiments. Moreover, we are monitoring the presence of specific dsRNA in non-transgenic leaves, sampled in different developmental stages. Although much remains to be elucidated about the trans-grafting technique, molecular and pathogen resistance analyses could indicate whether dsRNA is transferred from the producer HIGS rootstock to the combined non-transgenic scion.View Poster
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Lucía Del Castillo-Gonzálezhttps://doi.org/10.1094/ISMPMI-2023-26Phytophthora cinnamomi Rands (Pc), is considered one of the most virulent and invasive phytopathogens in the world, with a high incidence in non-forest and forest species such as chestnut, cork and holm oaks, avocado, blackberry, or tomato causing leaf and root rot, crown, and trunk canker. The life cycle of Pc involves the production of highly virulent mobile asexual zoospores (Zs), which allows their dispersion of them through soil and water currents by the aquatic environment, stablishing a high biodiversity of belowground interactions with several host. At this work we will show new approaches for the management of Zs under strictly controlled conditions, at the laboratory. Those techniques allowed us to simulate nature in the laboratory, for inoculating tomato seedlings with Zs. This poor studied pathosystem, allowed us to expand our current knowledge about the biodiversity of Pc's abilities to interact with plant host. View Poster
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Mamadou L. Fallhttps://doi.org/10.1094/ISMPMI-2023-27Grapevine leafroll-3 associated virus (GLRaV3) and grapevine Pinot gris virus (GPGV) are two RNA viruses of grapevine impacting significantly fruit yield and sugar content. Double-stranded RNAs (dsRNAs) were extracted from grapevine leaf samples of different plants collected in two sampling times (summer and autumn) over three years. cDNAs were sequenced using MiSeq Illumina. Bioinformatics analysis of datasets obtained from more than 200 samples was carried out using the viralrecon and iVar consensus reconstruction pipelines. The results indicate the presence of single nucleotide polymorphisms (SNPs) that gradually increased in both RNA viruses genomes. The SNP variations observed are diverse and implied the substitution of nitrogenous bases at the codon levels. For GLRaV3, some of these mutations appeared as early as the first year of planting and are conserved over time with some chimeric structural disruption of the p19.7 viral suppressor of RNA silencing protein, replicase protein, and replicase polyprotein. Whereas for GPGV, these mutations only started in the second year of planting and do not appear to have any impact at the protein level. Within the same plant, the nature of the mutations varied from one season to another and the symptomology followed the same seasonality. Overall, the results demonstrated that these two viruses can serve as an experimental model to study RNA virus evolution and the role of SNPs in fitness and symptom expression. View Poster
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Manuel Anguita-Maesohttps://doi.org/10.1094/ISMPMI-2023-28Currently, there is no culture medium described for axenic development of the olive xylem microbiota, which is critical for understanding the ecological interactions between vascular pathogens and native xylem-inhabiting bacteria. The purpose of this study was to characterize the effect of various broth culture media simulating xylem sap composition and the influence of olive genotype on the bacterial communities that emerge during culture. Xylem sap extracted from “Picual” and “Arbequina” olive cultivars were inoculated in SXM, XVM2, XF26, PD3, 3G10R and XDM2 broth culture media for 2 weeks. The increase in absorbance was monitored daily, and cell cultures aliquots were taken every 2 days to examine changes in bacterial community composition using NGS approach. Alpha and beta diversity indicated that the main differences were due to broth media used, followed by the olive xylem sap genotype, with a minor effect of incubation time. PD3 was the medium that best supported bacterial growth but enriched for the lowest bacterial ASVs; whereas XVM2 medium showed the highest ASVs detected in both genotypes, followed by 3G10R in “Picual” sap and XDM2 in “Arbequina” sap. However, whereas XVM2 and 3G10R supported well bacterial growth, XDM2 did not. The potential use of endotherapy treatments using enriched xylem microbiome cultures to modify indigenous xylem sap microbiome of olive plantlets will be discussed. View Poster
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Marcus Dillonhttps://doi.org/10.1094/ISMPMI-2023-29The bacterial genus Xanthomonas causes disease in several hundred plant species, many of which are economically important crops. In the era of next-generation sequencing, thousands of Xanthomonas genomes have now been sequenced as part of isolated studies that focus on outbreak characterization, host range, diversity, and virulence factor identification. However, these data have not been synthesized and we still lack a comprehensive phylogeny for the genus that reflects genome-wide relationships. Here, we present a pangenome analysis of 1,910 diverse Xanthomonas genomes, highlighting the extensive diversity in the genus, the evolutionary relationships between classified species, and the distribution of virulence factors across strains. We find a high degree of genetic cohesion that results from frequent horizontal gene transfer and identify a number of broadly conserved virulence factors. We also use these data to reassign incorrectly classified strains to phylogenetically informed species and find evidence of both monophyletic host-specificity and convergent evolution of distantly related strains to the same host. Understanding the evolutionary trajectory of host specificity in Xanthomonads and its relationship with key virulence factors provides valuable insight into the mechanisms through which these strains shift between hosts. In turn, this will enable us to develop more robust resistance strategies to protect important crops from these potentially devastating pathogens. View Poster
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Matthieu Joostenhttps://doi.org/10.1094/ISMPMI-2023-30Tomato leaf mold is caused by the fungus Fulvia fulva (syn. Cladosporium fulvum). Resistant tomato recognizes F. fulva avirulence (Avr) effectors by means of Cf receptor proteins, resulting in hypersensitive response-based resistance. So far, several Cf genes have been characterized and deployed, however the durability of these genes has proven to be limited. Durable resistance remains elusive since F. fulva readily overcomes Cf resistance by loss or mutation of the corresponding Avr effector, after which the deployed Cf gene becomes ineffective. To achieve a more durable form of genetic resistance, we are introgressing, mapping and characterizing a significant number of novel Cf genes from wild Solanum germplasm. In parallel, we are using CRISPR-Cas9 technology to generate knockouts of the corresponding effectors in F. fulva to determine their contribution to fungal virulence on susceptible tomato. We hypothesize that (combinations of) Cf genes corresponding to effectors that strongly contribute to virulence will result in more durable resistance. So far, we have taken steps towards mapping of the novel Cf genes, and we have generated knockouts of the corresponding effectors in F. fulva. Additionally, we have identified Avr9B matching the Cf-9 homolog, Cf-9B, explaining the sequential breakdown of the widely used Cf-9 resistance locus, and currently we are working on identifying Avr6, which matches the most recently deployed Cf protein, Cf-6. View Poster
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Meenu Singla-Rastogihttps://doi.org/10.1094/ISMPMI-2023-31We have recently reported that plants secrete diverse RNA species into the leaf apoplast, and that the great majority of this extracellular RNA (exRNA) is located outside of extracellular vesicles (EVs) with only a small proportion being intravesicular. This extravesicular exRNA is stabilized and protected against degradation from extracellular RNases, either through association with RNA-binding proteins (RPBs), formation of secondary structures (in the case of circular RNAs and tRNAs), or due to their post-transcriptional modifications. These observations indicate that intercellular exchange of RNAs may not require EVs. In our present study, we show that exRNA is not confined to the apoplast, but also reaches the leaf surface, probably through natural openings, such as stomata. Consistent with this hypothesis, the composition of leaf surface RNA is very similar to apoplastic RNA, comprising a heterogenous group of non-coding RNAs, with species as small as a dozen nucleotides to hundreds of nucleotides long. We have also observed an accumulation of exRNA upon PAMP elicitation. We will present our latest results on the role of these exRNAs in plant immune responses against different phytopathogens. View Poster
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Ning Zhanghttps://doi.org/10.1094/ISMPMI-2023-32Plant intracellular immune receptors, primarily nucleotide-binding, leucine-rich repeat proteins (NLRs), can detect virulence proteins (effectors) from pathogens and activate NLR-triggered immunity (NTI). Emerging evidence indicates that “sensor” NLRs often pair with “helper” NLRs to activate the immune response. We investigated the role of tomato helper NLRs, Nrc2 (NLR required for cell death 2) and Nrc3, on tomato immunity to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Loss-of-function mutations in both Nrc2 and Nrc3 (Dnrc2/3) completely compromised Pto/Prf-mediated NTI to Pst DC3000 containing the cognate effectors AvrPto and AvrPtoB. An nrc3 mutant exhibited susceptibility that was intermediate between wildtype plants and a prf mutant; an nrc2 mutant exhibited mild disease symptoms. These observations indicate that Nrc2 and Nrc3 contribute redundantly but also additively to Pto/Prf-mediated immunity to Pst. We also examined where Nrc2/3 act in the Pto/Prf-mediated immune response. In the Dnrc2/3 mutant, the hypersensitive response (HR) induced by overexpression of variants of AvrPtoB, Pto or Prf was compromised, but that induced by MKKKa or MKK2 was not. This indicates that Nrc2 and Nrc3 act upstream or independently of MAPK signaling and downstream of or together with Pto/Prf. Nrc2 and Nrc3 are not required for HR triggered by the sensor NLR Ptr1 in tomato, but appear to be involved in other NLR-mediated resistance against some Pst race 1 strains. View Poster
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Noor Azizah Musahttps://doi.org/10.1094/ISMPMI-2023-33The leguminous cover crop (LCC) is critical for oil palm plantations to reduce competition for nutrients from noxious weeds, to conserve soil moisture, and to improve soil fertility. However, the LCC can only efficiently fix the atmospheric nitrogen through a symbiotic association with Rhizobium bacteria into biologically available nitrogen to plants. This study assessed the influence of Rhizobium on a common LCC species i.e. Pueraria javanica (PJ), particularly in plant biomass, nodulation, nitrogen bioavailability in aboveground and belowground biomass, and microbial perpetuation in root nodules. Un-inoculated LCC seeds were tested as a control for the LCC seeds coated with Rhizobium. Interestingly, the growth of Rhizobium-inoculated PJ was significantly more vigorous in the form of plant biomass (51%), nodulation (37%), and nitrogen bioavailability in leaves (14%) and roots (15%). The findings can provide with a useful insight for the reduction in the application of chemical-based nitrogen fertilizer in oil palm plantations, especially at the juvenile stage. Besides, the assay method can also be extended to other LCC species such as Mucuna bracteates and Centrosema pubescens. View Poster
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Nutthalak Laksanavilathttps://doi.org/10.1094/ISMPMI-2023-34Blast disease caused by the fungus Magnaporthe oryzae is one of the most devastating rice diseases. To secure rice production from losses by blast, disease resistance genes are critical. Pi-ta is a widely used blast resistance gene. Published work reports that it codes for a nucleotide-binding and leucine-rich repeat protein (NLR) and recognizes the fungal protease-like effector AVR-Pita. However, this model was challenged by the recent finding that another rice gene named Ptr, which codes for a membrane protein with a cytoplasmic Armadillo repeat domain is required for AVR-Pita recognition. Using NLR Pi-ta and Ptr RNAi knock-down and CRIPSR-Cas9 mutant rice lines, we found that AVR-Pita recognition relies only on Ptr and that the NLR Pi-ta has no role. Analysis of the natural diversity of AVR-Pita showed that different alleles of Ptr have different recognition specificities. While the PtrB recognizes a restricted set of AVR-Pita alleles, PtrA seems to detect all natural sequence variants of the effector. We confirmed the escape of certain AVR-Pita alleles from detection by PtrB using mutant and transgenic isolates of the fungus and identified one specific polymorphism that controls the break-down of PtrB-mediated resistance. Taken together our work establishes that the M. oryzae effector AVR-Pita is detected in an allele specific manner by the unique rice resistance protein Ptr and that the NLR Pi-ta has no function in Pi-ta resistance and the recognition of AVR-Pita. View Poster
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Oseias Rodrigues Feitosa-Juniorhttps://doi.org/10.1094/ISMPMI-2023-35Xylella fastidiosa is an insect-transmitted bacterial pathogen able to colonize more than 600 plant species1 and causing severe diseases such as Olive Quick Decline Syndrome (OQDS)2,3. The ecological niche of X. fastidiosa is defined by the host plant's xylem vessels and the foregut of xylem sap-feeding insect vectors. The absence of a Type III Secretion System (T3SS)4 raises the question of whether the bacterium releases effectors to enable colonization. To address this, we used an adapted pipeline5 to predict secreted proteins from the pangenome of X. fastidiosa. We focused on candidate-secreted proteins that carry targeting signals for eukaryotic organelles, lack transmembrane domains, and are annotated as hypothetical proteins. This resulted in the prediction of 421 Xylella fastidiosa-secreted proteins (Xyp), including candidates Xyp1 to Xyp13 previously detected in the secretome6-9. Preliminary data suggest that Xyp1 can localize to plant nuclei. We will discuss the subcellular targeting of Xyp1 in plant cells and address its immunomodulatory potential. Homology modeling of the 421 Xyp protein structures revealed 5 clusters of proteins with similar structure models. Within these clusters, members are predicted to target diverse subcellular compartments. We, therefore, speculate that structurally-related Xyp candidates could have similar molecular functions, but in diverse locations within host cells. View Poster
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Paula Bellés Sanchohttps://doi.org/10.1094/ISMPMI-2023-36The beta-rhizobial strain Paraburkholderia phymatum STM815 can form a nitrogen-fixing symbiosis with the agriculturally important legume Phaseolus vulgaris. To investigate the role of the master regulator of nitrogen fixation NifA, we performed metabolomics and dual RNA-sequencing on common bean root nodules induced by P. phymatum wild-type and a non-fixing nifA mutant strain, previously reported to induce a higher nodule number and fused-nodule organization compared to wild-type strain. We discovered that P. phymatum NifA negatively regulates two genes (iaaM and iaaH) essential for auxin biosynthesis The mutation of iaaMH in a wild-type and nifA mutant background, revealed that P. phymatum produces indole-3-acetic acid (IAA) inside P. vulgaris nodules via the indole-acetamide (IAM) pathway. We demonstrate that NifA represses the expression of the iaaM and iaaH genes at all stages of the symbiosis by measuring their expression in the presence and absence of NifA. Moreover, we also examined the involvement of auxin in the nodule architecture of common bean nodules infected with the iaaMH and nifA mutants, showing that the iaaMH genes are responsible for the increased number of fused nodules in plants infected with the nifA mutant. Our data suggest a role for P. phymatum auxin production in the control of early-stage symbiotic interactions and that the increased abundance of rhizobial auxin in the nifA mutant strain enables greater root infection rates. View Poster
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Peter DiGennarohttps://doi.org/10.1094/ISMPMI-2023-37Root-knot nematodes (RKN; Meloidogyne spp.) establish and maintain specialized feeding cells within host tissue. This intimate interaction is likely regulated by monitoring host biology conserved across their extensive host range. One such conserved aspect of plant biology of temporal regulation of processes. RKN gene transcription and biology likely follow similar patterns. We profiled RKN and plant host Medicago truncatula gene transcription over time of mature parasite-host interactions and identified approximately 1,000 genes with variable trasncription levels, many of which are different during the dark (night) period. To characeterize the plant-nematode interaction over time, gene networks based on their expression level. This network highlighted the plant phenylpropanoid pathway. Further examination of this pathway confirmed that a similar pattern is present in other RKN hosts (tomato) through quantification of downstream phenylpropanoid pathway compounds, phenolics and flavonoids. This work elucidated novel interacting pathways that could be targeted to develop a RKN management strategy via plant tolerance or resistance. View Poster
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Poulami Sarkarhttps://doi.org/10.1094/ISMPMI-2023-38Citrus Huanglongbing (HLB), caused by Candidatus liberibacter asiaticus (CLas) is a serious disease of citrus inflicting huge economic losses in Florida. Most of the commercial citrus cultivars are susceptible to HLB, which calls for an effective disease management strategy with the development of HLB tolerant/ resistant cultivars. In susceptible varieties, strong activation of phloem defense responses leads to phloem collapse and reduction of phloem transport. Transgenic citrus expressing the Arabidopsis thaliana NPR1 (AtNPR1) protein displays enhanced tolerance against HLB. However, the mechanism underlying this tolerance is unknown. In this study, we analyzed the phloem defense responses in transgenic ‘Duncan’ grapefruit and ‘Hamlin’ sweet orange lines expressing AtNPR1. We discovered that, upon CLas infection, AtNPR1 lines had elevated levels of callose in the phloem but did not over-accumulate callose or ROS compared to the wild-type plants. Expression analysis of callose synthase genes, RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), and systemic acquired resistance-related genes validated these phenotypes. Furthermore, microscopy analysis revealed thicker phloem fibers and reduced phloem collapse in the AtNPR1 lines upon CLas infection. Our results suggest that the elevated basal immune responses in the AtNPR1 plants may suppress CLas-triggered robust immune responses seen in the wild-type plants, resulting in minimized symptom development and enhanced tolerance to CLas.View Poster
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Robin Cowperhttps://doi.org/10.1094/ISMPMI-2023-39Trichoderma are saprophytic fungi colonising plants. Some of them can promote plant growth, enhance tolerance to abiotic stress, and prime leaves for increased resistance to pathogen and insect attack. Nevertheless, when applied to different crops and under field conditions inconsistent beneficial effects are observed. Since Trichoderma’s plant-beneficial effects depend on efficient root colonisation, it is crucial to elucidate mechanisms involved in the recognition and colonisation of roots by Trichoderma. For this, we conducted a time-series RNAseq experiment to examine the transcriptional responses of Arabidopsis roots to Trichoderma colonisation and identify key factors regulating these responses. In vitro grown Arabidopsis seedlings were inoculated with the T. afroharzianum strain T22 and root samples were collected at 1, 2, 3 and 7 days. At 1 day after T22 colonisation, differential gene expression analysis showed a strong upregulation of jasmonic acid mediated defence signaling and GO term enrichment analysis suggested an important role of plant secondary metabolites (i.e., glucosinolates, camalexin, flavonoids). After 2 days of T22 colonisation an increase in salicylic acid signaling occurs, coinciding with increased expression of callose and lignin biosynthesis genes. These preliminary results indicate an interplay between plant hormones, secondary metabolites, and cell wall modifications during the early stages of root colonisation by T22. View Poster
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Roger Wisehttps://doi.org/10.1094/ISMPMI-2023-40Mildew resistance locus a (Mla) is essential to a gene interaction network that leads to life or death of the host. We used a dynamic time-course transcriptome of barley and the powdery mildew fungus, Blumeria hordei (Bh), to infer gene effects and epistatic relationships governed by the NLR encoded by Mla6, two other host loci critical to the interaction, Blufensin1 (Bln1) and Required for Mla6 resistance3 (Rar3), and the genes that interact with them. Bln1 is an R-gene independent regulator of immunity and the resistant bln1 mutant exhibits enhanced basal defense to compatible Bh. Rar3 is required for MLA6-mediated generation of H2O2 and the hypersensitive response; the susceptible rar3 mutant contains an in-frame Lys-Leu deletion in the SGS domain of SGT1. Gene effect models revealed epistatic interactions between Mla6 and Bln1, and the impact of Rar3 on the barley transcriptome. From a total of 468 annotated barley NLRs, 366 were expressed in our dataset and 115 of those were classified under different gene effect models, which clustered at several chromosome hotspots. The corresponding Bh infection transcriptome was classified into 9 co-expressed modules, linking differential expression with pathogen development. Expression of most of the 517 Bh effectors exhibited dependence on disease phenotype and was associated with appressorial or haustorial structures, suggesting that disease is regulated by a host-pathogen intercommunication network that diversifies the response. View Poster
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Sam T Mugfordhttps://doi.org/10.1094/ISMPMI-2023-41Sap-feeding insects of the order Hemiptera, such as aphids, whiteflies and leafhoppers, use stylets to navigate to the plant vascular system for long-term feeding. We previously demonstrated that aphids deposit the effector Mp10 into the cytoplasm of mesophyll cells. Mp10 belongs to a family of chemosensory proteins (CSPs) commonly present in arthropods. In aphids Mp10 is also known as CSP4. How CSPs regulate cellular processes in plants, and in eukaryotic cells in general, is largely unknown. We found that Mp10 suppresses PTI in response to a variety of elicitor molecules, including aphid-derived elicitors. Mp10 interacts with conserved plant target proteins that have key roles in the trafficking and stabilization of membrane proteins, in agreement with our data showing that Mp10 inhibits ROS and Ca2+ bursts to flg22 through the regulation of the pattern recognition receptor FLS2. Mining of available genome-sequence data revealed that Mp10 belongs to a clade shared with homologues from other plant-feeding hemipterans, and Mp10 orthologues from other sap-feeders share PTI-suppressive functions and interactions with plant targets. Thus, CSPs in the Mp10 clade may have evolved to suppress PTI in plants early in the evolution of plant-feeding hemipterans. Given that aphids deposit Mp10 in cells upon probing, this effector appears to act as a local anesthetic to inhibit plant perception of aphid feeding. View Poster
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Samuel Eastmanhttps://doi.org/10.1094/ISMPMI-2023-42Plant roots grow alongside a complex microbiome that includes both pathogenic and commensal bacteria. The plant immune system recognizes microbe-associated molecular patterns (MAMPs), including the flg22 epitope derived from bacterial flagellin, and reacts with a range of immune responses collectively termed MAMP-triggered immunity (MTI) that restrict microbial growth in the rhizosphere. Pathogenic bacteria suppress MTI by deploying virulence factors, most prominently effectors secreted through the type III secretion system. However, the regulation of MTI by commensal bacteria is not well understood. The commensal root bacteria Dyella japonica (Dja) MF79 suppresses MTI in a manner dependent on the type II, not type III secretion system. However, no type II secreted effectors responsible for MTI suppression have been identified. We analyzed Dja MF79 culture filtrates by mass spectrometry to identify secreted proteins with suppressive activity. We conducted targeted gene deletion of a candidate subtilisin-like serine protease we term Dyella immunosuppressive subtilase 1 (dis1). We assessed bacterial suppression of flg22-induced responses in transgenic Arabidopsis roots expressing a flg22-inducible GUS reporter inoculated with live bacteria or filtered culture supernatant. We observed that dis1 deletion reduces but does not completely eliminate MTI suppression by Dja MF79. Our results demonstrate Dis1 is a type II secreted effector that suppresses plant immunity. View Poster
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Samuel Eastmanhttps://doi.org/10.1094/ISMPMI-2023-43Toll/interleukin-1 receptor (TIR) domain proteins activate or suppress plant immunity by generating immunomodulatory purine nucleotides (IPNs) from NAD+. Plant TIR-domains convert NAD+ into a range of IPNs including ADPR and 2'cADPR while promoting immune activation and plant cell death. Conversely, the phytopathogenic TIR-domain effector HopAM1 converts NAD+ into 3'cADPR while suppressing plant immunity. However, it is still unclear if NAD+ depletion plays a significant role in TIR-mediated cell death caused by HopAM1 or plant TIR-domains. HopAM1 induces moderate accumulation of an unidentified molecule in Arabidopsis thaliana Col-0 and Xan-2. HopAM1’s TIR domain alone (HopAM1TIR) generates cADPR, ADPR, and an additional unidentified product in vitro rather than 3'cADPR. Pseudomonas fluorescens (pHIR11)-delivered HopA161 induces ADPR in Nicotiana tabacum cv. Xanthi but cADPR in A. thaliana Ws-0. HopAM1 suppresses production of both IPNs while blocking cell death. HopA161-induced cell death in N. tabacum cv. Xanthi correlates with production of IPNs, not with a depletion of NAD+. Finally, the IPN-binding phage protein Tad1 (Thoeris anti-defense 1) blocks HopAM1-induced cell death in yeast. Our results show considerably more complexity to TIR-domain enzymatic activity and indicate that NAD+-depletion is unlikely to be the primary mechanism of either HopAM1 or plant TIR domain-mediated cell death. View Poster
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Sandor Gondahttps://doi.org/10.1094/ISMPMI-2023-44The current study wishes to provide an insight on the complex interactions between fungal endophytes and the plant metabolites in a field setting. Data from 2-3-year field studies on horseradish were evaluated with inter-domain correlation analysis and path analysis (SEM). The metabolome was assessed by a quality-controlled, untargeted LC-MS approach. The endosphere fungal community was characterised with an amplicon-based metagenomics methdology. Chemically variable horseradish cultivars enabled ranking the plant metabolite classes according to their influence on the abundance of endophytic fungi. Fungal groups accounting for about 1/3 of total reads were significantly affected, including Agaricomycetes, Morosphaeriaceae, Capnodiales, Xylariales as well as the genera Setophoma, Tetracladium and Monosporascus. In addition to glucosinolates and downstream products, flavonoids and - suprisingly - many primary metabolites were found to be influential. Data on roots from an agricultural plot with diverse soils showed that the abunance of a fungal ASV in the soil rarely correlates with abundance in the endosphere and that various endosphere ASVs show distinct behaviors: the abundance of some is mainly driven by soil chemistry, that of others by the plant metabolome, abundance of various soil fungi, and so on. Our data highlight the complexity of the community assembly and the power of the untargeted metabolomics approach in studying plant-microbe interactions. View Poster
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SANDRA NNADIhttps://doi.org/10.1094/ISMPMI-2023-45Microbial communities inside and around the roots influence plant traits such as plant nutrition and growth, but traits such as flower development are rarely assessed in relation to the microbiome. We are examining the root fungal microbiome of the Northern Highbush blueberry (Vaccinium corymbosum) to determine whether there is a correlation between specific fungal taxa and floral traits such as flower number and bud formation. To test this, we inoculated blueberry plants with either commercial inoculum, native soil or a combination of both native soil and commercial inoculum. The plants were grown in pots for 3 years, and we harvested root samples twice each year. Using culture-independent methods, we observed that the 3-year-old plants before treatment already had a complex microbiome with the genus Oidiodendron having a high abundance (49%), but after treatment, its abundance reduced to 1% and did not persist after the first year. Serendipita, a fungal endophyte that can form ericoid mycorrhizae with roots of ericaceous plants was not observed in the plants prior to treatment but found in varying abundance after treatment. We also identified some specific taxa, Serendipita and Sarocladium, that correlated positively with bud number. In this way, we hope to provide new knowledge for developing strategies towards sustainable agricultural practices and set the stage for future understanding on how microbial interactions belowground influence aboveground traits. View Poster
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Sebastian Tonnhttps://doi.org/10.1094/ISMPMI-2023-46Compatible plant-pathogen interactions result in diverse visible plant disease symptoms. Assessing these symptoms is the basis for identifying plant disease and quantifying disease severity, such as in plant breeding programs for disease resistance. But already before the appearance of visible symptoms, the interaction has triggered fundamental changes in the plant. Optical sensors can non-invasively detect such changes, and therefore enable early disease detection and quantification as well as provide insights into processes underlying symptom development. This can be particularly useful for pathogens that induce visible symptoms only late in the disease cycle, for example obligate biotrophs like the oomycete downy mildews. We found that lettuce leaf tissue that is colonized by the downy mildew pathogen Bremia lactucae emits increased UV-A-excited blue-green fluorescence before the appearance of other visible signs and symptoms. Transcriptome and metabolome analysis suggest that this fluorescence originates mainly from the accumulation of phenolic acids, potential precursors for pathogen-induced lignin biosynthesis. We also show that UV-fluorescence imaging can be applied for early and accurate downy mildew disease severity estimation. In combination with automatic image segmentation, this can provide a tool for sensor-based phenotyping in downy mildew resistance breeding. View Poster
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Suchismita Ghoshhttps://doi.org/10.1094/ISMPMI-2023-47The hemibiotrophic fungus Colletotrichum initiates infection by penetrating host epidermal cells and forming biotrophic hyphae. We used Serial Block Face Scanning Electron Microscopy (SBF-SEM) to visually compare the infection progression between Medicago sativa and its adapted (compatible) pathogen Colletotrichum destructivum and non-adapted (incompatible) pathogen Colletotrichum higginsianum. Three-dimensional reconstruction of serial images using the software IMOD revealed that Medicago cotyledons infected with compatible C. destructivum undergo massive cytological changes to accommodate extensive fungal growth. Conversely, infection with incompatible C. higginsianum revealed fewer successful penetration events and frequent formation of papillary-like structures beneath attempted fungal penetration sites. Incompatible interaction sites often displayed vesicle-like structures uncommon in compatible interactions. We therefore examined the secretion of extracellular vesicles from Medicago plants infected with either compatible or incompatible Colletotrichum. We observed that at 60hrs post infection, there were significantly more vesicles secreted from Medicago infected with incompatible C. higginsianum compared to compatible C. destructivum, which in turn was more than produced by non-infected plants. Together, these results suggest that extracellular vesicles contribute to immunity during pathogen attack in M. sativa, possibly by mediating communication between them. View Poster
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TAN ANH NHI NGUYENhttps://doi.org/10.1094/ISMPMI-2023-48Endophytic fungus Colletotrichum tofieldiae (Ct) colonizes Arabidopsis thaliana roots and promotes plant growth via transferring phosphorus to the host under phosphorus deficiency. This requires the host’s tryptophan-derived metabolite biosynthetic pathway, as the defective cyp79B2 cyp79B3 mutant was killed by Ct. Our open field studies further implies that Ct can enhance plants growth in unfertilized fields, where soluble nitrogen sources are limited. Here, we employed a gnotobiotic agar system resembling field malnutrition status, where nitrate and/or ammonium contents were limited. Our results indicated that Ct promotes A. thaliana growth under both conditions where nitrate was the only nitrogen source and when nitrate was partially substituted by ammonium. RNA-seq analysis reveals that the root colonization by Ct strongly induced several A. thaliana nitrate or ammonium uptake genes. These results suggest that Ct root colonization helps plants to uptake nitrate and/or ammonium under nitrogen scarcity, which might be one of the keys to the growth promotion observed in fields. Interestingly, Ct supports cyp79B2 cyp79B3 mutant growth in this condition at the early time point, while gradually exhibiting antagonistic effects at the later stage. This may imply that the host's tryptophan-derived metabolites immunity is one of the critical factors that are required to maintain beneficial symbiosis and suppress Ct’s aggressiveness under nitrogen limitation. View Poster
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Tomohisa Shimasakihttps://doi.org/10.1094/ISMPMI-2023-49Plant specialized metabolites (PSMs) play a key role in interaction with root-associated microbial communities, called root microbiota, and genetic disruption of plant specialized metabolism alters the root microbiota taxonomic compositions. However, the molecular mechanisms by which root microbiota responds to PMSs remain unclear. We recently showed that Arthrobacter is one of the dominant taxa that characterize the root microbiota unique to tobacco plants, and Arthrobacter strains isolated from tobacco roots are capable of degrading tobacco-specific PSMs, such as nicotine. Here, we show that nicotine-catabolic genes in the Arthrobacter genus have been acquired multiple independent times via horizontal gene transfer events. Combined with experimental evolution analysis, we propose that the presence of nicotine in the inhabiting niche drives such horizontal acquisitions. Lastly, by performing a gnotobiotic inoculation experiment with a synthetic community (SynCom) with either a wild-type or a nicotine-catabolism mutant strain, we show that the catabolic capacity toward nicotine is crucial for the competitiveness of Arthrobacter in tobacco root, ultimately contributing to the assemblage of characteristic tobacco root microbiota. Our findings experimentally demonstrate the molecular mechanism by which PSMs and their catabolic genes in bacteria assemble root microbiota, and that mobile genetic elements facilitate the adaptation of root microbiota to host-specific environments. View Poster
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Willem E.W. Schravesandehttps://doi.org/10.1094/ISMPMI-2023-50Viral movement proteins (MPs) are critical for the local spread of viruses in plants by modulating plasmodesmata, membrane-lined pores connecting plant cells through cell walls. Lettuce Big-Vein associated Virus, a member of the Rhabdoviridae, has been linked to the Lettuce Big Vein Disease complex. This viral disease is characterized by vein clearing, banding, and retarded growth of lettuce plants. Despite that this virus is known for decades, its viral proteins have not been studied in detail yet. Using structural predictions made with AlphaFold2, we found that ORF3 of Lettuce Big-Vein associated Virus (LBVaV) appears to adopt the structural topology of the 30K-like movement protein family. This is remarkable, since classical protein multiple sequence alignment was unable to show such relationship. We next showed that ORF3 locates at the plasmodesmata by expressing it in concert with the marker protein PDCB1. To show that ORF3 from LBVaV facilitates cell-to-cell movement of plant viruses, movement-impaired infectious clones of Potato Virus X (PVX) and Tomato Mosaic Virus (ToMV) were co-expressed with LBVaV ORF3. LBVaV ORF3 indeed facilitated movement of both plant viruses. The combined observations revealed that LBVaV ORF3 encodes a 30K-like movement protein that facilitates viral cell-to-cell movement. View Poster
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William Underwoodhttps://doi.org/10.1094/ISMPMI-2023-51Basal stalk rot caused by the necrotrophic fungal pathogen Sclerotinia sclerotiorum is an economically important disease limiting sunflower production in the Northern Plains region of the US. S. sclerotiorum can infect a broad range of dicotyledonous plants and causes stem rot diseases on crops such as soybean and canola. In contrast, sunflower basal stalk rot begins as a root infection and is unique among Sclerotinia diseases of crop plants, which typically affect aerial tissues. Resistance to S. sclerotiorum is quantitative and is governed by many genes contributing small effects to the overall level of resistance. To identify loci contributing to basal stalk rot resistance and gain insights into molecular processes contributing to the defense response, we carried out genome-wide association mapping and RNAseq studies. Association mapping identified 19 loci associated with basal stalk rot resistance. In parallel, we compared transcriptional responses in root tissues of three susceptible and three partially resistant sunflower lines in a time-course after S. sclerotiorum inoculation. RNAseq results suggest that partially resistant lines exhibit distinct forms of resistance, and one resistant line appears to restrict entry into or colonization of root tissues at early stages of infection. These results provide important information to advance breeding efforts for improved basal stalk rot resistance and to further our understanding of quantitative resistance to this pathogen. View Poster
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Yoshiteru Noutoshihttps://doi.org/10.1094/ISMPMI-2023-52Chemical molecules that activate plant immunity are used in the field to protect crops from disease. From the screening of in-house chemically synthesized 1,600 cyclic peptides consisting of seven natural amino acids, we identified 10 candidates that enhanced Arabidopsis hypersensitive cell death of suspension cells triggered by an incompatible Pseudomonas syringae pv. tomato DC3000 avrRpm1. We evaluated the activity of three of them in inducing resistance to diseases caused by Rhizoctonia solani in Arabidopsis thaliana and Brachypodium distachyon. Pretreatment of detached Arabidopsis leaves with the cyclic peptides reduced the symptoms caused by R. solani AG-1 IC. Arabidopsis seedlings pretreated with the cyclic peptides also grew larger than the control in soil inoculated with R. solani AG-4. They also induced resistance in B. distachyon to R. solani AG-1 IA in leaf- and soil-inoculation assays. Pretreatment of Arabidopsis seedlings with the cyclic peptides accelerated the timing of MAPK activation and increased the production of reactive oxygen species in response to the flg22 peptide. Time-course transcriptome analysis of the cyclic peptide-treated Arabidopsis suspension cells revealed an upregulation of cell wall-associated genes and defense-related genes 6 h after the treatment. The identified cyclic peptides may be recognized as damage in the cell wall, thus priming plant immunity via the danger signal. This work was supported by JST ALCA and BRAIN. View Poster
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Zigmunds Orlovskishttps://doi.org/10.1094/ISMPMI-2023-53More than 92% of all vascular plant species on Earth form symbiotic associations with soil fungi, known as mycorrhizae, which extend the physiological surface of plant roots and provide plants with water and essential mineral nutrients. In addition, hyphae of arbuscular mycorrhizal fungi (AMF) can colonize and connect root systems of individual plants. Such underground network, known as common mycelial network (CMN), is proposed to function as a super-organism or information highway in transferring diverse signals from plant-to-plant in response to biotic stress stimuli. To address this hypothesis, we utilize a model AMF Rhizophagus irregularis to colonize and connect Medicago truncatula plants as well as Daucus carota hairy root cultures and explore the effect of known defence elicitors, including flg22, and wounding on the expression of plant defence marker genes and pathogen performance in the systemic leaves of donor plants or AMF-connected inter-plant signal receivers. Our preliminary data suggest that AMF-colonization status modulates intra-plant responses to flg22 and wounding compared to AMF-free plants and potentially mediates inter-plant signals that could act in plant defence and pathogen resistance. View Poster
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