Leaf cuticular wax composition of a genetically diverse collection of lettuce (Lactuca sativa L.) cultivars evaluated under field conditions.

Cuticular waxes of plants impart tolerance to many forms of environmental stress and help shed dangerous human pathogens on edible plant parts. Although the chemical composition of waxes on a wide variety of important crops has been described, a detailed wax compositional analysis has yet to be reported for lettuce (Lactuca sativa L.), one of the most widely consumed vegetables. We present herein the leaf wax content and composition of 12 genetically diverse lettuce cultivars sampled across five time points during their vegetative growth phase in the field. Mean total leaf wax amounts across all cultivars varied little over 28 days of vegetative growth, except for a notable decrease in total waxes following a major precipitation event, presumably due to wax degradation from wind and rain. All lettuce cultivars were found to contain a unique wax composition highly enriched in 22- and 24-carbon length 1-alcohols (docosanol and tetracosanol, respectively). In our report, the dominance of these shorter chain length 1-alcohols as wax constituents represents a relatively rare phenotype in plants. The ecological significance of these dominant and relatively short 1-alcohols is still unknown. Although waxes have been a target for improvement of various crops, no such work has been reported for lettuce. This study lays the groundwork for future research that aims to integrate cuticular wax characteristics of field grown plants into the larger context of lettuce breeding and cultivar development.

Genome assembly and analysis of Lactuca virosa: implications for lettuce breeding.

Lettuce (Lactuca sativa L.) is a leafy vegetable crop with ongoing breeding efforts related to quality, resilience, and innovative production systems. To breed resilient and resistant lettuce in the future, valuable genetic variation found in close relatives could be further exploited. Lactuca virosa (2x = 2n = 18), a wild relative assigned to the tertiary lettuce gene pool, has a much larger genome (3.7 Gbp) than Lactuca sativa (2.5 Gbp). It has been used in interspecific crosses and is a donor to modern crisphead lettuce cultivars. Here, we present a de novo reference assembly of L. virosa with high continuity and complete gene space. This assembly facilitated comparisons to the genome of L. sativa and to that of the wild species L. saligna, a representative of the secondary lettuce gene pool. To assess the diversity in gene content, we classified the genes of the 3 Lactuca species as core, accessory, and unique. In addition, we identified 3 interspecific chromosomal inversions compared to L. sativa, which each may cause recombination suppression and thus hamper future introgression breeding. Using 3-way comparisons in both reference-based and reference-free manners, we show that the proliferation of long-terminal repeat elements has driven the genome expansion of L. virosa. Further, we performed a genome-wide comparison of immune genes, nucleotide-binding leucine-rich repeat, and receptor-like kinases among Lactuca spp. and indicated the evolutionary patterns and mechanisms behind their expansions. These genome analyses greatly facilitate the understanding of genetic variation in L. virosa, which is beneficial for the breeding of improved lettuce varieties.

Contrasting allelic effects for pistachio salinity tolerance in juvenile and mature trees.

Breeding perennial tree crops often requires prediction of mature performance from juvenile data. To assess the utility of juvenile screens to predict salinity tolerance of mature pistachio trees, we compared performance of 3-month ungrafted seedlings and 4-year-old grafted rootstocks under salinity stress. The QTL allele associated with higher salt exclusion from seedling leaves conferred lower growth in saline field conditions, suggesting that mapping QTL in seedlings may be easier than discerning the optimal allele for field performance.

Genome-Enabled Insights into Downy Mildew Biology and Evolution.

Oomycetes that cause downy mildew diseases are highly specialized, obligately biotrophic phytopathogens that can have major impacts on agriculture and natural ecosystems. Deciphering the genome sequence of these organisms provides foundational tools to study and deploy control strategies against downy mildew pathogens (DMPs). The recent telomere-to-telomere genome assembly of the DMP Peronospora effusa revealed high levels of synteny with distantly related DMPs, higher than expected repeat content, and previously undescribed architectures. This provides a road map for generating similar high-quality genome assemblies for other oomycetes. This review discusses biological insights made using this and other assemblies, including ancestral chromosome architecture, modes of sexual and asexual variation, the occurrence of heterokaryosis, candidate gene identification, functional validation, and population dynamics. We also discuss future avenues of research likely to be fruitful in studies of DMPs and highlight resources necessary for advancing our understanding and ability to forecast and control disease outbreaks.

PhytoOracle: Scalable, modular phenomics data processing pipelines.

As phenomics data volume and dimensionality increase due to advancements in sensor technology, there is an urgent need to develop and implement scalable data processing pipelines. Current phenomics data processing pipelines lack modularity, extensibility, and processing distribution across sensor modalities and phenotyping platforms. To address these challenges, we developed PhytoOracle (PO), a suite of modular, scalable pipelines for processing large volumes of field phenomics RGB, thermal, PSII chlorophyll fluorescence 2D images, and 3D point clouds. PhytoOracle aims to (i) improve data processing efficiency; (ii) provide an extensible, reproducible computing framework; and (iii) enable data fusion of multi-modal phenomics data. PhytoOracle integrates open-source distributed computing frameworks for parallel processing on high-performance computing, cloud, and local computing environments. Each pipeline component is available as a standalone container, providing transferability, extensibility, and reproducibility. The PO pipeline extracts and associates individual plant traits across sensor modalities and collection time points, representing a unique multi-system approach to addressing the genotype-phenotype gap. To date, PO supports lettuce and sorghum phenotypic trait extraction, with a goal of widening the range of supported species in the future. At the maximum number of cores tested in this study (1,024 cores), PO processing times were: 235 minutes for 9,270 RGB images (140.7 GB), 235 minutes for 9,270 thermal images (5.4 GB), and 13 minutes for 39,678 PSII images (86.2 GB). These processing times represent end-to-end processing, from raw data to fully processed numerical phenotypic trait data. Repeatability values of 0.39-0.95 (bounding area), 0.81-0.95 (axis-aligned bounding volume), 0.79-0.94 (oriented bounding volume), 0.83-0.95 (plant height), and 0.81-0.95 (number of points) were observed in Field Scanalyzer data. We also show the ability of PO to process drone data with a repeatability of 0.55-0.95 (bounding area).

The genome of Lactuca saligna, a wild relative of lettuce, provides insight into non-host resistance to the downy mildew Bremia lactucae.

Lactuca saligna L. is a wild relative of cultivated lettuce (Lactuca sativa L.), with which it is partially interfertile. Hybrid progeny suffer from hybrid incompatibility (HI), resulting in reduced fertility and distorted transmission ratios. Lactuca saligna displays broad-spectrum resistance against lettuce downy mildew caused by Bremia lactucae Regel and is considered a non-host species. This phenomenon of resistance in L. saligna is called non-host resistance (NHR). One possible mechanism behind this NHR is through the plant-pathogen interaction triggered by pathogen recognition receptors, including nucleotide-binding leucine-rich repeat (NLR) proteins and receptor-like kinases (RLKs). We report a chromosome-level genome assembly of L. saligna (accession CGN05327), leading to the identification of two large paracentric inversions (>50 Mb) between L. saligna and L. sativa. Genome-wide searches delineated the major resistance clusters as regions enriched in NLRs and RLKs. Three of the enriched regions co-locate with previously identified NHR intervals. RNA-seq analysis of Bremia-infected lettuce identified several differentially expressed RLKs in NHR regions. Three tandem wall-associated kinase-encoding genes (WAKs) in the NHR8 interval display particularly high expression changes at an early stage of infection. We propose RLKs as strong candidates for determinants of the NHR phenotype of L. saligna.

EffectorO: Motif-Independent Prediction of Effectors in Oomycete Genomes Using Machine Learning and Lineage Specificity.

Oomycete plant pathogens cause a wide variety of diseases, including late blight of potato, sudden oak death, and downy mildews of plants. These pathogens are major contributors to loss in numerous food crops. Oomycetes secrete effector proteins to manipulate their hosts to the advantage of the pathogen. Plants have evolved to recognize effectors, resulting in an evolutionary cycle of defense and counter-defense in plant-microbe interactions. This selective pressure results in highly diverse effector sequences that can be difficult to computationally identify using only sequence similarity. We developed a novel effector prediction tool, EffectorO, that uses two complementary approaches to predict effectors in oomycete pathogen genomes: i) a machine learning-based pipeline that predicts effector probability based on the biochemical properties of the N-terminal amino-acid sequence of a protein and ii) a pipeline based on lineage specificity to find proteins that are unique to one species or genus, a sign of evolutionary divergence due to adaptation to the host. We tested EffectorO on Bremia lactucae, which causes lettuce downy mildew, and Phytophthora infestans, which causes late blight of potato and tomato, and predicted many novel effector candidates while recovering the majority of known effector candidates. EffectorO will be useful for discovering novel families of oomycete effectors without relying on sequence similarity to known effectors. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Whole-genome sequence of synthetically derived Brassica napus inbred cultivar Da-Ae.

Brassica napus, a globally important oilseed crop, is an allotetraploid hybrid species with two subgenomes originating from Brassica rapa and Brassica oleracea. The presence of two highly similar subgenomes has made the assembly of a complete draft genome challenging and has also resulted in natural homoeologous exchanges between the genomes, resulting in variations in gene copy number, which further complicates assigning sequences to correct chromosomes. Despite these challenges, high-quality draft genomes of this species have been released. Using third generation sequencing and assembly technologies, we generated a new genome assembly for the synthetic B. napus cultivar Da-Ae. Through the use of long reads, linked-reads, and Hi-C proximity data, we assembled a new draft genome that provides a high-quality reference genome of a synthetic B. napus. In addition, we identified potential hotspots of homoeologous exchange between subgenomes within Da-Ae, based on their presence in other independently derived lines. The occurrence of these hotspots may provide insight into the genetic rearrangements required for B. napus to be viable following the hybridization of B. rapa and B. oleracea.

The genome of the oomycete Peronosclerospora sorghi, a cosmopolitan pathogen of maize and sorghum, is inflated with dispersed pseudogenes.

Several species in the oomycete genus Peronosclerospora cause downy mildew on maize and can result in significant yield losses in Asia. Bio-surveillance of these pathogens is a high priority to prevent epidemics on maize in the United States and consequent damage to the US economy. The unresolved taxonomy and dearth of molecular resources for Peronosclerospora spp. hinder these efforts. P. sorghi is a pathogen of sorghum and maize with a global distribution, for which limited diversity has been detected in the southern USA. We characterized the genome, transcriptome, and mitogenome of an isolate, representing the US pathotype 6. The highly homozygous genome was assembled using 10× Genomics linked reads and scaffolded using Hi-C into 13 chromosomes. The total assembled length was 303.2 Mb, larger than any other oomycete previously assembled. The mitogenome was 38 kb, similar in size to other oomycetes, although it had a unique gene order. Nearly 20,000 genes were annotated in the nuclear genome, more than described for other downy mildew causing oomycetes. The 13 chromosomes of P. sorghi were highly syntenic with the 17 chromosomes of Peronospora effusa with conserved centromeric regions and distinct chromosomal fusions. The increased assembly size and gene count of P. sorghi is due to extensive retrotransposition, resulting in putative pseudogenization. Ancestral genes had higher transcript abundance and were enriched for differential expression. This study provides foundational resources for analysis of Peronosclerospora and comparisons to other oomycete genera. Further genomic studies of global Peronosclerospora spp. will determine the suitability of the mitogenome, ancestral genes, and putative pseudogenes for marker development and taxonomic relationships.

GRF-GIF chimeric proteins enhance in vitro regeneration and Agrobacterium-mediated transformation efficiencies of lettuce (Lactuca spp.).

KEY MESSAGE: GRF-GIF chimeric proteins from multiple source species enhance in vitro regeneration in both wild and cultivated lettuce. In addition, they enhance regeneration in multiple types of lettuce including butterheads, romaines, and crispheads. The ability of plants to regenerate in vitro has been exploited for use in tissue culture systems for plant propagation, plant transformation, and genome editing. The success of in vitro regeneration is often genotype dependent and continues to be a bottleneck for Agrobacterium-mediated transformation and its deployment for improvement of some crop species. Manipulation of transcription factors that play key roles in plant development such as BABY BOOM, WUSCHEL, and GROWTH-REGULATING FACTORs (GRFs) has improved regeneration and transformation efficiencies in several plant species. Here, we compare the efficacy of GRF-GIF gene fusions from multiple species to boost regeneration efficiency and shooting frequency in four genotypes of wild and cultivated lettuce (Lactuca spp. L.). In addition, we show that GRF-GIFs with mutated miRNA 396 binding sites increase regeneration efficiency and shooting frequency when compared to controls. We also present a co-transformation strategy for increased transformation efficiency and recovery of transgenic plants harboring a gene of interest. This strategy will enhance the recovery of transgenic plants of other lettuce genotypes and likely other crops in the Compositae family.

Pistachio genomes provide insights into nut tree domestication and ZW sex chromosome evolution.

Pistachio is a nut crop domesticated in the Fertile Crescent and a dioecious species with ZW sex chromosomes. We sequenced the genomes of Pistacia vera cultivar (cv.) Siirt, the female parent, and P. vera cv. Bagyolu, the male parent. Two chromosome-level reference genomes of pistachio were generated, and Z and W chromosomes were assembled. The ZW chromosomes originated from an autosome following the first inversion, which occurred approximately 8.18 Mya. Three inversion events in the W chromosome led to the formation of a 12.7-Mb (22.8% of the W chromosome) non-recombining region. These W-specific sequences contain several genes of interest that may have played a pivotal role in sex determination and contributed to the initiation and evolution of a ZW sex chromosome system in pistachio. The W-specific genes, including defA, defA-like, DYT1, two PTEN1, and two tandem duplications of six VPS13A paralogs, are strong candidates for sex determination or differentiation. Demographic history analysis of resequenced genomes suggest that cultivated pistachio underwent severe domestication bottlenecks approximately 7640 years ago, dating the domestication event close to the archeological record of pistachio domestication in Iran. We identified 390, 211, and 290 potential selective sweeps in 3 cultivar subgroups that underlie agronomic traits such as nut development and quality, grafting success, flowering time shift, and drought tolerance. These findings have improved our understanding of the genomic basis of sex determination/differentiation and horticulturally important traits and will accelerate the improvement of pistachio cultivars and rootstocks.

Genome assembly and association tests identify interacting loci associated with vigor, precocity, and sex in interspecific pistachio rootstocks.

Understanding the basis of hybrid vigor remains a key question in crop breeding and improvement, especially for rootstock development where F1 hybrids are extensively utilized. Full-sibling UCB-1 F1 seedling rootstocks are widely planted in commercial pistachio orchards that are generated by crossing 2 highly heterozygous outbreeding parental trees of Pistacia atlantica (female) and P. integerrima (male). This results in extensive phenotypic variability, prompting costly removal of low-yielding small trees. To identify the genetic basis of this variability, we assembled chromosome-scale genome assemblies of the parental trees of UCB-1. We genotyped 960 UCB-1 trees in an experimental orchard for which we also collected multiyear phenotypes. We genotyped an additional 1,358 rootstocks in 6 commercial pistachio orchards and collected single-year tree-size data. Genome-wide single marker association tests identified loci associated with tree size and shape, sex, and precocity. In the experimental orchard, we identified multiple trait-associated loci and a strong candidate for ZZ/ZW sex chromosomes. We found significant marker associations unique to different traits and to early vs late phenotypic measures of the same trait. We detected 2 loci strongly associated with rootstock size in commercial orchards. Pseudo-testcross classification of markers demonstrated that the trait-associated alleles for each locus were segregating in the gametes of opposite parents. These 2 loci interact epistatically to generate the bimodal distribution of tree size with undesirable small trees observed by growers. We identified candidate genes within these regions. These findings provide a foundational resource for marker development and genetic selection of vigorous pistachio UCB-1 rootstock.

Loss-of-function of SAWTOOTH 1 affects leaf dorsiventrality genes to promote leafy heads in lettuce.

The mechanisms underlying leafy heads in vegetables are poorly understood. Here, we cloned a quantitative trait locus (QTL) controlling leafy heads in lettuce (Lactuca sativa). The QTL encodes a transcription factor, SAWTOOTH 1 (LsSAW1), which has a BEL1-like homeodomain and is a homolog of Arabidopsis thaliana. A 1-bp deletion in Lssaw1 contributes to the development of leafy heads. Laser-capture microdissection and RNA-sequencing showed that LsSAW1 regulates leaf dorsiventrality and loss-of-function of Lssaw1 downregulates the expression of many adaxial genes but upregulates abaxial genes. LsSAW1 binds to the promoter region of the adaxial gene ASYMMETRIC LEAVES 1 (LsAS1) to upregulate its expression. Overexpression of LsAS1 compromised the effects of Lssaw1 on heading. LsSAW1 also binds to the promoter region of the abaxial gene YABBY 1 (LsYAB1), but downregulates its expression. Overexpression of LsYAB1 led to bending leaves in LsSAW1 genotypes. LsSAW1 directly interacts with KNOTTED 1 (LsKN1), which is necessary for leafy heads in lettuce. RNA-seq data showed that LsSAW1 and LsKN1 exert antagonistic effects on the expression of thousands of genes. LsSAW1 compromises the ability of LsKN1 to repress LsAS1. Our results suggest that downregulation or loss-of-function of adaxial genes and upregulation of abaxial genes allow for the development of leafy heads.

Identification of genetic loci in lettuce mediating quantitative resistance to fungal pathogens.

KEY MESSAGE: We demonstrate genetic variation for quantitative resistance against important fungal pathogens in lettuce and its wild relatives, map loci conferring resistance and predict key molecular mechanisms using transcriptome profiling. Lactuca sativa L. (lettuce) is an important leafy vegetable crop grown and consumed globally. Chemicals are routinely used to control major pathogens, including the causal agents of grey mould (Botrytis cinerea) and lettuce drop (Sclerotinia sclerotiorum). With increasing prevalence of pathogen resistance to fungicides and environmental concerns, there is an urgent need to identify sources of genetic resistance to B. cinerea and S. sclerotiorum in lettuce. We demonstrated genetic variation for quantitative resistance to B. cinerea and S. sclerotiorum in a set of 97 diverse lettuce and wild relative accessions, and between the parents of lettuce mapping populations. Transcriptome profiling across multiple lettuce accessions enabled us to identify genes with expression correlated with resistance, predicting the importance of post-transcriptional gene regulation in the lettuce defence response. We identified five genetic loci influencing quantitative resistance in a F6 mapping population derived from a Lactuca serriola (wild relative) × lettuce cross, which each explained 5-10% of the variation. Differential gene expression analysis between the parent lines, and integration of data on correlation of gene expression and resistance in the diversity set, highlighted potential causal genes underlying the quantitative trait loci.

Molecular Determinants of in vitro Plant Regeneration: Prospects for Enhanced Manipulation of Lettuce (Lactuca sativa L.).

In vitro plant regeneration involves dedifferentiation and molecular reprogramming of cells in order to regenerate whole organs. Plant regeneration can occur via two pathways, de novo organogenesis and somatic embryogenesis. Both pathways involve intricate molecular mechanisms and crosstalk between auxin and cytokinin signaling. Molecular determinants of both pathways have been studied in detail in model species, but little is known about the molecular mechanisms controlling de novo shoot organogenesis in lettuce. This review provides a synopsis of our current knowledge on molecular determinants of de novo organogenesis and somatic embryogenesis with an emphasis on the former as well as provides insights into applying this information for enhanced in vitro regeneration in non-model species such as lettuce (Lactuca sativa L.).

The upregulated LsKN1 gene transforms pinnately to palmately lobed leaves through auxin, gibberellin, and leaf dorsiventrality pathways in lettuce.

Leaf shape represents a vital agronomic trait for leafy vegetables such as lettuce. Some lettuce cultivars produce lobed leaves, varying from pinnately to palmately lobed, but the genetic mechanisms remain unclear. In this study, we cloned one major quantitative trait locus (QTL) controlling palmately lobed leaves. The candidate gene, LsKN1, encodes a homeobox transcription factor, and has been shown previously to be critical for the development of leafy heads in lettuce. The LsKN1 allele that is upregulated by the insertion of a transposon promotes the development of palmately lobed leaves. We demonstrated that LsKN1 upregulated LsCUC2 and LsCUC3 through different mechanisms, and their upregulation was critical for the development of palmately lobed leaves. LsKN1 binds the promoter of LsPID to promote auxin biosynthesis, which positively contributes to the development of palmately lobed leaves. In contrast, LsKN1 suppresses GA biosynthesis to promote palmately lobed leaves. LsKN1 also binds to the promoter of LsAS1, a dorsiventrality gene, to downregulate its expression. Overexpression of the LsAS1 gene compromised the effects of the LsKN1 gene changing palmately to pinnately lobed leaves. Our study illustrated that the upregulated LsKN1 gene led to palmately lobed leaves in lettuce by integrating several downstream pathways, including auxin, gibberellin, and leaf dorsiventrality pathways.

Variance of allele balance calculated from low coverage sequencing data infers departure from a diploid state.

BACKGROUND: Polyploidy and heterokaryosis are common and consequential genetic phenomena that increase the number of haplotypes in an organism and complicate whole-genome sequence analysis. Allele balance has been used to infer polyploidy and heterokaryosis in diverse organisms using read sets sequenced to greater than 50× whole-genome coverage. However, sequencing to adequate depth is costly if applied to multiple individuals or large genomes. RESULTS: We developed VCFvariance.pl to utilize the variance of allele balance to infer polyploidy and/or heterokaryosis at low sequence coverage. This analysis requires as little as 10× whole-genome coverage and reduces the allele balance profile down to a single value, which can be used to determine if an individual has two or more haplotypes. This approach was validated using simulated, synthetic, and authentic read sets from the oomycete species Bremia lactucae and Phytophthora infestans, the fungal species Saccharomyces cerevisiae, and the plant species Arabidopsis arenosa. This approach was deployed to determine that nine of 21 genotyped European race-type isolates of Bremia lactucae were inconsistent with diploidy and therefore likely heterokaryotic. CONCLUSIONS: Variance of allele balance is a reliable metric to detect departures from a diploid state, including polyploidy, heterokaryosis, a mixed sample, or chromosomal copy number variation. Deploying this strategy is computationally inexpensive, can reduce the cost of sequencing by up to 80%, and used to test any organism.

Viral Load Among Vaccinated and Unvaccinated, Asymptomatic and Symptomatic Persons Infected With the SARS-CoV-2 Delta Variant.

We found no significant difference in cycle threshold values between vaccinated and unvaccinated persons infected with severe acute respiratory syndrome coronavirus 2 Delta, overall or stratified by symptoms. Given the substantial proportion of asymptomatic vaccine breakthrough cases with high viral levels, interventions, including masking and testing, should be considered in settings with elevated coronavirus disease 2019 transmission.

Ancestral Chromosomes for Family Peronosporaceae Inferred from a Telomere-to-Telomere Genome Assembly of Peronospora effusa.

Downy mildew disease of spinach, caused by the oomycete Peronospora effusa, causes major losses to spinach production. In this study, the 17 chromosomes of P. effusa were assembled telomere-to-telomere, using Pacific Biosciences high-fidelity reads. Of these, 16 chromosomes are complete and gapless; chromosome 15 contains one gap bridging the nucleolus organizer region. This is the first telomere-to-telomere genome assembly for an oomycete. Putative centromeric regions were identified on all chromosomes. This new assembly enables a reevaluation of the genomic composition of Peronospora spp.; the assembly was almost double the size and contained more repeat sequences than previously reported for any Peronospora species. Genome fragments consistently underrepresented in six previously reported assemblies of P. effusa typically encoded repeats. Some genes annotated as encoding effectors were organized into multigene clusters on several chromosomes. Putative effectors were annotated on 16 of the 17 chromosomes. The intergenic distances between annotated genes were consistent with compartmentalization of the genome into gene-dense and gene-sparse regions. Genes encoding putative effectors were enriched in gene-sparse regions. The near-gapless assembly revealed apparent horizontal gene transfer from Ascomycete fungi. Gene order was highly conserved between P. effusa and the genetically oriented assembly of the oomycete Bremia lactucae; high levels of synteny were also detected with Phytophthora sojae. Extensive synteny between phylogenetically distant species suggests that many other oomycete species may have similar chromosome organization. Therefore, this assembly provides the foundation for genomic analyses of diverse oomycetes.[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.

Pseudomonas syringae effector HopZ3 suppresses the bacterial AvrPto1-tomato PTO immune complex via acetylation.

The plant pathogen Pseudomonas syringae secretes multiple effectors that modulate plant defenses. Some effectors trigger defenses due to specific recognition by plant immune complexes, whereas others can suppress the resulting immune responses. The HopZ3 effector of P. syringae pv. syringae B728a (PsyB728a) is an acetyltransferase that modifies not only components of plant immune complexes, but also the Psy effectors that activate these complexes. In Arabidopsis, HopZ3 acetylates the host RPM1 complex and the Psy effectors AvrRpm1 and AvrB3. This study focuses on the role of HopZ3 during tomato infection. In Psy-resistant tomato, the main immune complex includes PRF and PTO, a RIPK-family kinase that recognizes the AvrPto effector. HopZ3 acts as a virulence factor on tomato by suppressing AvrPto1Psy-triggered immunity. HopZ3 acetylates AvrPto1Psy and the host proteins PTO, SlRIPK and SlRIN4s. Biochemical reconstruction and site-directed mutagenesis experiments suggest that acetylation acts in multiple ways to suppress immune signaling in tomato. First, acetylation disrupts the critical AvrPto1Psy-PTO interaction needed to initiate the immune response. Unmodified residues at the binding interface of both proteins and at other residues needed for binding are acetylated. Second, acetylation occurs at residues important for AvrPto1Psy function but not for binding to PTO. Finally, acetylation reduces specific phosphorylations needed for promoting the immune-inducing activity of HopZ3's targets such as AvrPto1Psy and PTO. In some cases, acetylation competes with phosphorylation. HopZ3-mediated acetylation suppresses the kinase activity of SlRIPK and the phosphorylation of its SlRIN4 substrate previously implicated in PTO-signaling. Thus, HopZ3 disrupts the functions of multiple immune components and the effectors that trigger them, leading to increased susceptibility to infection. Finally, mass spectrometry used to map specific acetylated residues confirmed HopZ3's unusual capacity to modify histidine in addition to serine, threonine and lysine residues.