AvrSr27 is a zinc-bound effector with a modular structure important for immune recognition.

Effector proteins are central to the success of plant pathogens, while immunity in host plants is driven by receptor-mediated recognition of these effectors. Understanding the molecular details of effector-receptor interactions is key for the engineering of novel immune receptors. Here, we experimentally determined the crystal structure of the Puccinia graminis f. sp. tritici (Pgt) effector AvrSr27, which was not accurately predicted using AlphaFold2. We characterised the role of the conserved cysteine residues in AvrSr27 using in vitro biochemical assays and examined Sr27-mediated recognition using transient expression in Nicotiana spp. and wheat protoplasts. The AvrSr27 structure contains a novel ß-strand rich modular fold consisting of two structurally similar domains that bind to Zn2+ ions. The N-terminal domain of AvrSr27 is sufficient for interaction with Sr27 and triggering cell death. We identified two Pgt proteins structurally related to AvrSr27 but with low sequence identity that can also associate with Sr27, albeit more weakly. Though only the full-length proteins, trigger Sr27-dependent cell death in transient expression systems. Collectively, our findings have important implications for utilising protein prediction platforms for effector proteins, and those embarking on bespoke engineering of immunity receptors as solutions to plant disease.

Pooled effector library screening in protoplasts rapidly identifies novel Avr genes.

Crop breeding for durable disease resistance is challenging due to the rapid evolution of pathogen virulence. While progress in resistance (R) gene cloning and stacking has accelerated in recent years1-3, the identification of corresponding avirulence (Avr) genes in many pathogens is hampered by the lack of high-throughput screening options. To address this technology gap, we developed a platform for pooled library screening in plant protoplasts to allow rapid identification of interacting R-Avr pairs. We validated this platform by isolating known and novel Avr genes from wheat stem rust (Puccinia graminis f. sp. tritici) after screening a designed library of putative effectors against individual R genes. Rapid Avr gene identification provides molecular tools to understand and track pathogen virulence evolution via genotype surveillance, which in turn will lead to optimized R gene stacking and deployment strategies. This platform should be broadly applicable to many crop pathogens and could potentially be adapted for screening genes involved in other protoplast-selectable traits.

Virulence Patterns of Oat Crown Rust in Australia - Season 2022.

Puccinia coronata f. sp. avenae (Pca) is an important foliar pathogen of oat which causes crown rust disease. The virulence profile of 48 Pca isolates derived from different locations in Australia was characterized using a collection of oat lines often utilized in rust surveys in the United States and Australia. This analysis indicates that Pca populations in Eastern Australia are broadly virulent, which contrasts with the population in Western Australia (WA). Several oat lines/Pc genes are effective against all rust samples collected from WA, suggesting they may provide useful resistance in this region if deployed in combination. We identified 19 lines from the United States oat differential set that display disease resistance to Pca in WA, with some in agreement with previous rust survey reports. We adopted the 10-letter nomenclature system to define oat crown rust races in Australia and compare the frequency of those virulence traits to published data from the United States. Based on this nomenclature, 42 unique races were detected among the 48 isolates, reflecting the high diversity of virulence phenotypes for Pca in Australia. Nevertheless, the Pca population in the United States is substantially more broadly virulent than that of Australia. Close examination of resistance profiles for the oat differential set lines after infection with Pca supports hypotheses of allelism or redundancy among Pc genes or the presence of several resistance genes in some oat differential lines. These findings illustrate the need to deconvolute the oat differential set using molecular tools.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome-Enabled Analysis of Population Dynamics and Virulence-Associated Loci in the Oat Crown Rust Fungus Puccinia coronata f. sp. avenae.

Puccinia coronata f. sp. avenae (Pca) is an important fungal pathogen causing crown rust that impacts oat production worldwide. Genetic resistance for crop protection against Pca is often overcome by the rapid virulence evolution of the pathogen. This study investigated the factors shaping adaptive evolution of Pca using pathogen populations from distinct geographic regions within the United States and South Africa. Phenotypic and genome-wide sequencing data of these diverse Pca collections, including 217 isolates, uncovered phylogenetic relationships and established distinct genetic composition between populations from northern and southern regions from the United States and South Africa. The population dynamics of Pca involve a bidirectional movement of inoculum between northern and southern regions of the United States and contributions from clonality and sexuality. The population from South Africa is solely clonal. A genome-wide association study (GWAS) employing a haplotype-resolved Pca reference genome was used to define 11 virulence-associated loci corresponding to 25 oat differential lines. These regions were screened to determine candidate Avr effector genes. Overall, the GWAS results allowed us to identify the underlying genetic factors controlling pathogen recognition in an oat differential set used in the United States to assign pathogen races (pathotypes). Key GWAS findings support complex genetic interactions in several oat lines, suggesting allelism among resistance genes or redundancy of genes included in the differential set, multiple resistance genes recognizing genetically linked Avr effector genes, or potentially epistatic relationships. A careful evaluation of the composition of the oat differential set accompanied by the development or implementation of molecular markers is recommended. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genotypic and Resistance Profile Analysis of Two Oat Crown Rust Differential Sets Urge Coordination and Standardization.

Puccinia coronata f. sp. avenae is the causal agent of the disease known as crown rust, which represents a bottleneck in oat production worldwide. Characterization of pathogen populations often involves race (pathotype) assignments using differential sets, which are not uniform across countries. This study compared the virulence profiles of 25 P. coronata f. sp. avenae isolates from Australia using two host differential sets, one from Australia and one from the United States. These differential sets were also genotyped using diversity arrays technology sequencing technology. Phenotypic and genotypic discrepancies were detected on 8 out of 29 common lines between the two sets, indicating that pathogen race assignments based on those lines are not comparable. To further investigate molecular markers that could assist in the stacking of rust resistance genes important for Australia, four published Pc91-linked markers were validated across the differential sets and then screened across a collection of 150 oat cultivars. Drover, Aladdin, and Volta were identified as putative carriers of the Pc91 locus. This is the first report to confirm that the cultivar Volta carries Pc91 and demonstrates the value of implementing molecular markers to characterize materials in breeding pools of oat. Overall, our findings highlight the necessity of examining seed stocks using pedigree and molecular markers to ensure seed uniformity and bring robustness to surveillance methodologies. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Nuclear exchange generates population diversity in the wheat leaf rust pathogen Puccinia triticina.

In clonally reproducing dikaryotic rust fungi, non-sexual processes such as somatic nuclear exchange are postulated to play a role in diversity but have been difficult to detect due to the lack of genome resolution between the two haploid nuclei. We examined three nuclear-phased genome assemblies of Puccinia triticina, which causes wheat leaf rust disease. We found that the most recently emerged Australian lineage was derived by nuclear exchange between two pre-existing lineages, which originated in Europe and North America. Haplotype-specific phylogenetic analysis reveals that repeated somatic exchange events have shuffled haploid nuclei between long-term clonal lineages, leading to a global P. triticina population representing different combinations of a limited number of haploid genomes. Thus, nuclear exchange seems to be the predominant mechanism generating diversity and the emergence of new strains in this otherwise clonal pathogen. Such genomics-accelerated surveillance of pathogen evolution paves the way for more accurate global disease monitoring.

Arbuscular mycorrhizal fungi heterokaryons have two nuclear populations with distinct roles in host-plant interactions.

Arbuscular mycorrhizal fungi (AMF) are prominent root symbionts that can carry thousands of nuclei deriving from two parental strains in a large syncytium. These co-existing genomes can also vary in abundance with changing environmental conditions. Here we assemble the nuclear genomes of all four publicly available AMF heterokaryons using PacBio high-fidelity and Hi-C sequencing. We find that the two co-existing genomes of these strains are phylogenetically related but differ in structure, content and epigenetics. We confirm that AMF heterokaryon genomes vary in relative abundance across conditions and show this can lead to nucleus-specific differences in expression during interactions with plants. Population analyses also reveal signatures of genetic exchange indicative of past events of sexual reproduction in these strains. This work uncovers the origin and contribution of two nuclear genomes in AMF heterokaryons and opens avenues for the improvement and environmental application of these strains.

A highly contiguous genome assembly reveals sources of genomic novelty in the symbiotic fungus Rhizophagus irregularis.

The root systems of most plant species are aided by the soil-foraging capacities of symbiotic arbuscular mycorrhizal (AM) fungi of the Glomeromycotina subphylum. Despite recent advances in our knowledge of the ecology and molecular biology of this mutualistic symbiosis, our understanding of the AM fungi genome biology is just emerging. Presented here is a close to T2T genome assembly of the model AM fungus Rhizophagus irregularis DAOM197198, achieved through Nanopore long-read DNA sequencing and Hi-C data. This haploid genome assembly of R. irregularis, alongside short- and long-read RNA-Sequencing data, was used to produce a comprehensive annotation catalog of gene models, repetitive elements, small RNA loci, and DNA cytosine methylome. A phylostratigraphic gene age inference framework revealed that the birth of genes associated with nutrient transporter activity and transmembrane ion transport systems predates the emergence of Glomeromycotina. While nutrient cycling in AM fungi relies on genes that existed in ancestor lineages, a burst of Glomeromycotina-restricted genetic innovation is also detected. Analysis of the chromosomal distribution of genetic and epigenetic features highlights evolutionarily young genomic regions that produce abundant small RNAs, suggesting active RNA-based monitoring of genetic sequences surrounding recently evolved genes. This chromosome-scale view of the genome of an AM fungus genome reveals previously unexplored sources of genomic novelty in an organism evolving under an obligate symbiotic life cycle.

Haplotype-phased and chromosome-level genome assembly of Puccinia polysora, a giga-scale fungal pathogen causing southern corn rust.

Rust fungi are characterized by large genomes with high repeat content and have two haploid nuclei in most life stages, which makes achieving high-quality genome assemblies challenging. Here, we described a pipeline using HiFi reads and Hi-C data to assemble a gigabase-sized fungal pathogen, Puccinia polysora f.sp. zeae, to haplotype-phased and chromosome-scale. The final assembled genome is 1.71 Gbp, with ~850 Mbp and 18 chromosomes in each haplotype, being currently one of the two giga-scale fungi assembled to chromosome level. Transcript-based annotation identified 47,512 genes for the dikaryotic genome with a similar number for each haplotype. A high level of interhaplotype variation was found with 10% haplotype-specific BUSCO genes, 5.8 SNPs/kbp, and structural variation accounting for 3% of the genome size. The P. polysora genome displayed over 85% repeat contents, with genome-size expansion and copy number increasing of species-specific orthogroups. Interestingly, these features did not affect overall synteny with other Puccinia species having smaller genomes. Fine-time-point transcriptomics revealed seven clusters of coexpressed secreted proteins that are conserved between two haplotypes. The fact that candidate effectors interspersed with all genes indicated the absence of a "two-speed genome" evolution in P. polysora. Genome resequencing of 79 additional isolates revealed a clonal population structure of P. polysora in China with low geographic differentiation. Nevertheless, a minor population differentiated from the major population by having mutations on secreted proteins including AvrRppC, indicating the ongoing virulence to evade recognition by RppC, a major resistance gene in Chinese corn cultivars. The high-quality assembly provides valuable genomic resources for future studies on disease management and the evolution of P. polysora.

A chromosome-level, fully phased genome assembly of the oat crown rust fungus Puccinia coronata f. sp. avenae: a resource to enable comparative genomics in the cereal rusts.

Advances in sequencing technologies as well as development of algorithms and workflows have made it possible to generate fully phased genome references for organisms with nonhaploid genomes such as dikaryotic rust fungi. To enable discovery of pathogen effectors and further our understanding of virulence evolution, we generated a chromosome-scale assembly for each of the 2 nuclear genomes of the oat crown rust pathogen, Puccinia coronata f. sp. avenae (Pca). This resource complements 2 previously released partially phased genome references of Pca, which display virulence traits absent in the isolate of historic race 203 (isolate Pca203) which was selected for this genome project. A fully phased, chromosome-level reference for Pca203 was generated using PacBio reads and Hi-C data and a recently developed pipeline named NuclearPhaser for phase assignment of contigs and phase switch correction. With 18 chromosomes in each haplotype and a total size of 208.10 Mbp, Pca203 has the same number of chromosomes as other cereal rust fungi such as Puccinia graminis f. sp. tritici and Puccinia triticina, the causal agents of wheat stem rust and wheat leaf rust, respectively. The Pca203 reference marks the third fully phased chromosome-level assembly of a cereal rust to date. Here, we demonstrate that the chromosomes of these 3 Puccinia species are syntenous and that chromosomal size variations are primarily due to differences in repeat element content.

Seeing is believing: Exploiting advances in structural biology to understand and engineer plant immunity.

Filamentous plant pathogens cause disease in numerous economically important crops. These pathogens secrete virulence proteins, termed effectors, that modulate host cellular processes and promote infection. Plants have evolved immunity receptors that detect effectors and activate defence pathways, resulting in resistance to the invading pathogen. This leads to an evolutionary arms race between pathogen and host that is characterised by highly diverse effector repertoires in plant pathogens. Here, we review the recent advances in understanding host-pathogen co-evolution provided by the structural determination of effectors alone, and in complex with immunity receptors. We highlight the use of recent advances in structural prediction within this field and its role for future development of designer resistance proteins.

Physical separation of haplotypes in dikaryons allows benchmarking of phasing accuracy in Nanopore and HiFi assemblies with Hi-C data.

BACKGROUND: Most animals and plants have more than one set of chromosomes and package these haplotypes into a single nucleus within each cell. In contrast, many fungal species carry multiple haploid nuclei per cell. Rust fungi are such species with two nuclei (karyons) that contain a full set of haploid chromosomes each. The physical separation of haplotypes in dikaryons means that, unlike in diploids, Hi-C chromatin contacts between haplotypes are false-positive signals. RESULTS: We generate the first chromosome-scale, fully-phased assembly for the dikaryotic leaf rust fungus Puccinia triticina and compare Nanopore MinION and PacBio HiFi sequence-based assemblies. We show that false-positive Hi-C contacts between haplotypes are predominantly caused by phase switches rather than by collapsed regions or Hi-C read mis-mappings. We introduce a method for phasing of dikaryotic genomes into the two haplotypes using Hi-C contact graphs, including a phase switch correction step. In the HiFi assembly, relatively few phase switches occur, and these are predominantly located at haplotig boundaries and can be readily corrected. In contrast, phase switches are widespread throughout the Nanopore assembly. We show that haploid genome read coverage of 30-40 times using HiFi sequencing is required for phasing of the leaf rust genome, with 0.7% heterozygosity, and that HiFi sequencing resolves genomic regions with low heterozygosity that are otherwise collapsed in the Nanopore assembly. CONCLUSIONS: This first Hi-C based phasing pipeline for dikaryons and comparison of long-read sequencing technologies will inform future genome assembly and haplotype phasing projects in other non-haploid organisms.

Long reads and Hi-C sequencing illuminate the two-compartment genome of the model arbuscular mycorrhizal symbiont Rhizophagus irregularis.

Chromosome folding links genome structure with gene function by generating distinct nuclear compartments and topologically associating domains. In mammals, these undergo preferential interactions and regulate gene expression. However, their role in fungal genome biology is unclear. Here, we combine Nanopore (ONT) sequencing with chromatin conformation capture sequencing (Hi-C) to reveal chromosome and epigenetic diversity in a group of obligate plant symbionts: the arbuscular mycorrhizal fungi (AMF). We find that five phylogenetically distinct strains of the model AMF Rhizophagus irregularis carry 33 chromosomes with substantial within-species variability in size, as well as in gene and repeat content. Strain-specific Hi-C contact maps reveal a 'checkerboard' pattern that underline two dominant euchromatin (A) and heterochromatin (B) compartments. Each compartment differs in the level of gene transcription, regulation of candidate effectors and methylation frequencies. The A-compartment is more gene-dense and contains most core genes, while the B-compartment is more repeat-rich and has higher rates of chromosomal rearrangement. While the B-compartment is transcriptionally repressed, it has significantly more secreted proteins and in planta upregulated candidate effectors, suggesting a possible host-induced change in chromosome conformation. Overall, this study provides a fine-scale view into the genome biology and evolution of model plant symbionts, and opens avenues to study the epigenetic mechanisms that modify chromosome folding during host-microbe interactions.

EffectorP 3.0: Prediction of Apoplastic and Cytoplasmic Effectors in Fungi and Oomycetes.

Many fungi and oomycete species are devasting plant pathogens. These eukaryotic filamentous pathogens secrete effector proteins to facilitate plant infection. Fungi and oomycete pathogens have diverse infection strategies and their effectors generally do not share sequence homology. However, they occupy similar host environments, either the plant apoplast or plant cytoplasm, and, therefore, may share some unifying properties based on the requirements of these host compartments. Here, we exploit these biological signals and present the first classifier (EffectorP 3.0) that uses two machine-learning models: one trained on apoplastic effectors and one trained on cytoplasmic effectors. EffectorP 3.0 accurately predicts known apoplastic and cytoplasmic effectors in fungal and oomycete secretomes with low estimated false-positive rates of 3 and 8%, respectively. Cytoplasmic effectors have a higher proportion of positively charged amino acids, whereas apoplastic effectors are enriched for cysteine residues. The combination of fungal and oomycete effectors in training leads to a higher number of predicted cytoplasmic effectors in biotrophic fungi. EffectorP 3.0 expands predicted effector repertoires beyond small, cysteine-rich secreted proteins in fungi and RxLR-motif containing secreted proteins in oomycetes. We show that signal peptide prediction is essential for accurate effector prediction, because EffectorP 3.0 recognizes a cytoplasmic signal also in intracellular, nonsecreted proteins.[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.

The stem rust fungus Puccinia graminis f. sp. tritici induces centromeric small RNAs during late infection that are associated with genome-wide DNA methylation.

BACKGROUND: Silencing of transposable elements (TEs) is essential for maintaining genome stability. Plants use small RNAs (sRNAs) to direct DNA methylation to TEs (RNA-directed DNA methylation; RdDM). Similar mechanisms of epigenetic silencing in the fungal kingdom have remained elusive. RESULTS: We use sRNA sequencing and methylation data to gain insight into epigenetics in the dikaryotic fungus Puccinia graminis f. sp. tritici (Pgt), which causes the devastating stem rust disease on wheat. We use Hi-C data to define the Pgt centromeres and show that they are repeat-rich regions (~250 kb) that are highly diverse in sequence between haplotypes and, like in plants, are enriched for young TEs. DNA cytosine methylation is particularly active at centromeres but also associated with genome-wide control of young TE insertions. Strikingly, over 90% of Pgt sRNAs and several RNAi genes are differentially expressed during infection. Pgt induces waves of functionally diversified sRNAs during infection. The early wave sRNAs are predominantly 21 nts with a 5' uracil derived from genes. In contrast, the late wave sRNAs are mainly 22-nt sRNAs with a 5' adenine and are strongly induced from centromeric regions. TEs that overlap with late wave sRNAs are more likely to be methylated, both inside and outside the centromeres, and methylated TEs exhibit a silencing effect on nearby genes. CONCLUSIONS: We conclude that rust fungi use an epigenetic silencing pathway that might have similarity with RdDM in plants. The Pgt RNAi machinery and sRNAs are under tight temporal control throughout infection and might ensure genome stability during sporulation.

Genomics accelerated isolation of a new stem rust avirulence gene-wheat resistance gene pair.

Stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt) is a devastating disease of the global staple crop wheat. Although this disease was largely controlled in the latter half of the twentieth century, new virulent strains of Pgt, such as Ug99, have recently evolved1,2. These strains have caused notable losses worldwide and their continued spread threatens global wheat production. Breeding for disease resistance provides the most cost-effective control of wheat rust diseases3. A number of rust resistance genes have been characterized in wheat and most encode immune receptors of the nucleotide-binding leucine-rich repeat (NLR) class4, which recognize pathogen effector proteins known as avirulence (Avr) proteins5. However, only two Avr genes have been identified in Pgt so far, AvrSr35 and AvrSr50 (refs. 6,7), and none in other cereal rusts8,9. The Sr27 resistance gene was first identified in a wheat line carrying an introgression of the 3R chromosome from Imperial rye10. Although not deployed widely in wheat, Sr27 is widespread in the artificial crop species Triticosecale (triticale), which is a wheat-rye hybrid and is a host for Pgt11,12. Sr27 is effective against Ug99 (ref. 13) and other recent Pgt strains14,15. Here, we identify both the Sr27 gene in wheat and the corresponding AvrSr27 gene in Pgt and show that virulence to Sr27 can arise experimentally and in the field through deletion mutations, copy number variation and expression level polymorphisms at the AvrSr27 locus.

The crystal structure of SnTox3 from the necrotrophic fungus Parastagonospora nodorum reveals a unique effector fold and provides insight into Snn3 recognition and pro-domain protease processing of fungal effectors.

Plant pathogens cause disease through secreted effector proteins, which act to promote infection. Typically, the sequences of effectors provide little functional information and further targeted experimentation is required. Here, we utilized a structure/function approach to study SnTox3, an effector from the necrotrophic fungal pathogen Parastagonospora nodorum, which causes cell death in wheat-lines carrying the sensitivity gene Snn3. We developed a workflow for the production of SnTox3 in a heterologous host that enabled crystal structure determination and functional studies. We show this approach can be successfully applied to study effectors from other pathogenic fungi. The ß-barrel fold of SnTox3 is a novel fold among fungal effectors. Structure-guided mutagenesis enabled the identification of residues required for Snn3 recognition. SnTox3 is a pre-pro-protein, and the pro-domain of SnTox3 can be cleaved in vitro by the protease Kex2. Complementing this, an in silico study uncovered the prevalence of a conserved motif (LxxR) in an expanded set of putative pro-domain-containing fungal effectors, some of which can be cleaved by Kex2 in vitro. Our in vitro and in silico study suggests that Kex2-processed pro-domain (designated here as K2PP) effectors are common in fungi and this may have broad implications for the approaches used to study their functions.

Austropuccinia psidii, causing myrtle rust, has a gigabase-sized genome shaped by transposable elements.

Austropuccinia psidii, originating in South America, is a globally invasive fungal plant pathogen that causes rust disease on Myrtaceae. Several biotypes are recognized, with the most widely distributed pandemic biotype spreading throughout the Asia-Pacific and Oceania regions over the last decade. Austropuccinia psidii has a broad host range with more than 480 myrtaceous species. Since first detected in Australia in 2010, the pathogen has caused the near extinction of at least three species and negatively affected commercial production of several Myrtaceae. To enable molecular and evolutionary studies into A. psidii pathogenicity, we assembled a highly contiguous genome for the pandemic biotype. With an estimated haploid genome size of just over 1 Gb (gigabases), it is the largest assembled fungal genome to date. The genome has undergone massive expansion via distinct transposable element (TE) bursts. Over 90% of the genome is covered by TEs predominantly belonging to the Gypsy superfamily. These TE bursts have likely been followed by deamination events of methylated cytosines to silence the repetitive elements. This in turn led to the depletion of CpG sites in TEs and a very low overall GC content of 33.8%. Compared to other Pucciniales, the intergenic distances are increased by an order of magnitude indicating a general insertion of TEs between genes. Overall, we show how TEs shaped the genome evolution of A. psidii and provide a greatly needed resource for strategic approaches to combat disease spread.

A functional genomics approach to dissect spotted alfalfa aphid resistance in Medicago truncatula.

Aphids are virus-spreading insect pests affecting crops worldwide and their fast population build-up and insecticide resistance make them problematic to control. Here, we aim to understand the molecular basis of spotted alfalfa aphid (SAA) or Therioaphis trifolii f. maculata resistance in Medicago truncatula, a model organism for legume species. We compared susceptible and resistant near isogenic Medicago lines upon SAA feeding via transcriptome sequencing. Expression of genes involved in defense and stress responses, protein kinase activity and DNA binding were enriched in the resistant line. Potentially underlying some of these changes in gene expression was the finding that members of the MYB, NAC, AP2 domain and ERF transcription factor gene families were differentially expressed in the resistant versus susceptible lines. A TILLING population created in the resistant cultivar was screened using exome capture sequencing and served as a reverse genetics tool to functionally characterise genes involved in the aphid resistance response. This screening revealed three transcription factors (a NAC, AP2 domain and ERF) as important regulators in the defence response, as a premature stop-codon in the resistant background led to a delay in aphid mortality and enhanced plant susceptibility. This combined functional genomics approach will facilitate the future development of pest resistant crops by uncovering candidate target genes that can convey enhanced aphid resistance.