Transgenic and gene-editing technologies are essential for gene functional analysis and crop improvement. However, the pleiotropic effects and unknown mechanisms of morphogenic genes have hindered their broader application. In this study, we employed the one-step de novo shoot organogenesis (DNSO) method, and demonstrated that overexpression of the morphogenic gene Arabidopsis thanalia GROWTH-REGULATING FACTOR 5 (AtGRF5) significantly enhanced genetic transformation efficiency in cucurbit crops by promoting callus proliferation and increasing dense cells during regeneration. High-resolution time-series transcriptomics and single-cell RNA sequencing revealed that AtGRF5 overexpression induced auxin-related genes and expanded stem cell populations during cucumber DNSO. Using DNA-affinity purification sequencing (DAP-seq) in combination with spatiotemporal differential gene expression analysis, we identified CsIAA19 as a key downstream target of AtGRF5, with its modulation playing a pivotal role in regeneration. Rescuing CsIAA19 in AtGRF5-overexpressing explant reversed the enhanced callus proliferation and regeneration. To address growth defects caused by AtGRF5 overexpression, we developed an abscisic acid-inducible AtGRF5 expression system, significantly improving transformation and gene-editing efficiency across diverse genotypes while minimizing pleiotropic effects. In summary, this research provides mechanistic insights into AtGRF5-mediated transformation and offers a practical solution to overcome challenges in cucurbit crop genetic modification.

Saline–alkaline soils are a major environmental problem that limit plant growth and crop productivity. Plasma membrane H⁺-ATPases and the salt overly sensitive (SOS) signaling pathway play important roles in plant responses to saline–alkali stress. However, little is known about the functional genes and mechanisms regulating the transcription of H⁺-ATPases and SOS pathway genes under saline–alkali stress. In the present study, we identified that the plant AT-rich sequence and zinc-binding (TaPLATZ2) transcription factor are involved in wheat response to saline–alkali stress by directly suppressing the expression of TaHA2/TaSOS3. The knockdown of TaPLATZ2 enhances salt and alkali stress tolerance, while overexpression of TaPLATZ2 leads to salt and alkali stress sensitivity in wheat. In addition, TaWRKY55 directly upregulated the expression of TaPLATZ2 during saline–alkali stress. Through knockdown and overexpression of TaWRKY55 in wheat, TaWRKY55 was shown to negatively modulate salt and alkali stress tolerance. Genetic analyses confirmed that TaPLATZ2 functions downstream of TaWRKY55 in response to salt and alkaline stresses. These findings provide a TaWRKY55–TaPLATZ2–TaHA2/TaSOS3 regulatory module that regulates wheat responses to saline–alkali stress.

Pineapple is the third most crucial tropical fruit worldwide and available in five varieties. Genomes of different pineapple varieties have been released to date; however, none of them are complete, with all exhibiting substantial gaps and representing only two of the five pineapple varieties. This significantly hinders the advancement of pineapple breeding efforts. In this study, we sequenced the genomes of three varieties: a wild pineapple variety, a fiber pineapple variety, and a globally cultivated edible pineapple variety. We constructed the first gap-free reference genome (Ref) for pineapple. By consolidating multiple sources of evidence and manually revising each gene structure annotation, we identified 26,656 protein-coding genes. The BUSCO evaluation indicated a completeness of 99.2%, demonstrating the high quality of the gene structure annotations in this genome. Utilizing these resources, we identified 7,209 structural variations across the three varieties. Approximately 30.8% of pineapple genes were located within ±5 kb of structural variations, including 30 genes associated with anthocyanin synthesis. Further analysis and functional experiments demonstrated that the high expression of AcMYB528 aligns with the accumulation of anthocyanins in the leaves, both of which may be affected by a 1.9-kb insertion fragment. In addition, we developed the Ananas Genome Database, which offers data browsing, retrieval, analysis, and download functions. The construction of this database addresses the lack of pineapple genome resource databases. In summary, we acquired a seamless pineapple reference genome with high-quality gene structure annotations, providing a solid foundation for pineapple genomics and a valuable reference for pineapple breeding.

Rice stripe mosaic virus (RSMV) is an emerging pathogen which significantly reduces rice yields in the southern region of China. It is transmitted by the leafhopper Recilia dorsalis, which overwinters in rice fields. Our field investigations revealed that RSMV infection causes delayed rice heading, resulting in a large number of green diseased plants remaining in winter rice fields. This creates a favorable environment for leafhoppers and viruses to overwinter, potentially contributing to the rapid spread and epidemic of the disease. Next, we explored the mechanism by which RSMV manipulates the developmental processes of the rice plant. A rice heading‐related E3 ubiquitin ligase, Heading date Associated Factor 1 (HAF1), was found to be hijacked by the RSMV‐encoded P6. The impairment of HAF1 function affects the ubiquitination and degradation of downstream proteins, HEADING DATE 1 and EARLY FLOWERING3, leading to a delay in rice heading. Our results provide new insights into the development regulation‐based molecular interactions between virus and plant, and highlights the importance of understanding virus‐vector‐plant tripartite interactions for effective disease management strategies.

Lodging reduces grain yield and quality in cereal crops. Lodging resistance is affected by the strength of the culm, which is influenced by the culm diameter, culm wall thickness, and cell wall composition. To explore the genetic architecture of culm diameter in rice (Oryza sativa), we conducted a genome-wide association study (GWAS). We identified STRONG CULM 2 (STRONG2), which encodes the mannan synthase CSLA5, and showed that plants that overexpressed this gene had increased culm diameter and improved lodging resistance. STRONG2 appears to increase the levels of cell wall components, such as mannose and cellulose, thereby enhancing sclerenchyma development in stems. SNP14931253 in the STRONG2 promoter contributes to variation in STRONG2 expression in natural germplasms and the transcription factor MYB61 directly activates STRONG2 expression. Furthermore, STRONG2 overexpressing plants produced significantly more grains per panicle and heavier grains than the wild-type plants. These results demonstrate that the MYB61–STRONG2 module positively regulates culm diameter and lodging resistance, information that could guide breeding efforts for improved yield in rice.

Glycosylation, a prevalent post-translational modification in eukaryotic secreted and membrane-associated proteins, plays a pivotal role in diverse physiological and pathological processes. Although UDP-N-acetylglucosamine (UDP-GlcNAc) is essential for this modification, the specific glycosylation mechanisms during plant leaf senescence and defense responses remain poorly understood. In our research, we identified a novel rice mutant named rbb1 (resistance to blast and bacterial blight1), exhibiting broad-spectrum disease resistance. This mutant phenotype results from a loss-of-function mutation in the gene encoding glucosamine-6-phosphate acetyltransferase, an important enzyme in D-glucosamine 6-phosphate acetylation. The rbb1 mutant demonstrates enhanced defense responses, evident in increased resistance to rice blast and bacterial blight, along with the upregulation of defense-response genes. Various biochemical markers indicate an activated defense mechanism in the rbb1 mutant, such as elevated levels of reactive oxygen species and malondialdehyde, reduced enzyme activity and UDP-GlcNAc content, and decreased expression of N-glycan and O-glycan modifying proteins. Moreover, proteome analysis of N-glycosylation modifications reveals alterations in the N-glycosylation of several disease-resistance-related proteins, with a significant reduction in Prx4 and Prx13 in rbb1-1. Additionally, the knockout of Prx4 or Prx13 also enhances resistance to Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae (M. oryzae). This study uncovers a novel mechanism of defense response in rice, suggesting potential targets for the development of disease-resistant varieties.

Coniferous forests are under severe threat of the rapid anthropogenic climate warming. Abies (firs), the fourth-largest conifer genus, is a keystone component of the boreal and temperate dark-coniferous forests and harbors a remarkably large number of relict taxa. However, the uncertainty of the phylogenetic and biogeographic history of Abies significantly impedes our prediction of future dynamics and efficient conservation of firs. In this study, using 1,533 nuclear genes generated from transcriptome sequencing and a complete sampling of all widely recognized species, we have successfully reconstructed a robust phylogeny of global firs, in which four clades are strongly supported and all intersectional relationships are resolved, although phylogenetic discordance caused mainly by incomplete lineage sorting and hybridization was detected. Molecular dating and ancestral area reconstruction suggest a Northern Hemisphere high-latitude origin of Abies during the Late Cretaceous, but all extant firs diversified during the Miocene to the Pleistocene, and multiple continental and intercontinental dispersals took place in response to the late Neogene climate cooling and orogenic movements. Notably, four critically endangered firs endemic to subtropical mountains of China, including A. beshanzuensis, A. ziyuanensis, A. fanjingshanensis and A. yuanbaoshanensis from east to west, have different origins and evolutionary histories. Moreover, three hotspots of species richness, including western North America, central Japan, and the Hengduan Mountains, were identified in Abies. Elevation and precipitation, particularly precipitation of the coldest quarter, are the most significant environmental factors driving the global distribution pattern of fir species diversity. Some morphological traits are evolutionarily constrained, and those linked to elevational variation (e.g., purple cone) and cold resistance (e.g., pubescent branch and resinous bud) may have contributed to the diversification of global firs. Our study sheds new light on the spatiotemporal evolution of global firs, which will be of great help to forest management and species conservation in a warming world.

Synthetic microbial communities (SynComs) are a promising tool for making full use of the beneficial functions imparted by whole bacterial consortia. However, the complexity of reconstructed SynComs often limits their application in sustainable agriculture. Furthermore, inter-strain interactions are often neglected during SynCom construction. Here, we propose a strategy for constructing a simplified and functional SynCom (sfSynCom) by using elite helper strains that significantly improve the beneficial functions of the core symbiotic strain, here Bradyrhizobium elkanii BXYD3, to sustain the growth of soybean (Glycine max). We first identified helper strains that significantly promote nodulation and nitrogen fixation in soybean mediated by BXYD3. Two of these helper strains assigned to the Pantoea taxon produce acyl homoserine lactones, which significantly enhanced the colonization and infection of soybean by BXYD3. Finally, we constructed a sfSynCom from these core and helper strains. This sfSynCom based on the core-helper strategy was more effective at promoting nodulation than inoculation with BXYD3 alone and achieved effects comparable to those of a complex elite SynCom previously constructed on the basis of potential beneficial functions between microbes and plants alone. Our results suggest that considering interactions between strains as well as those between strains and the host plant might allow construction of sfSynComs.

In C₃ plants, photorespiration is an energy expensive pathway that competes with photosynthetic CO₂ assimilation and releases CO₂ into the atmosphere, potentially reducing C₃ plant productivity by 20%-50%. Consequently, reducing the flux through photorespiration has been recognized as a major way to improve C₃ crop photosynthetic carbon fixation and productivity. While current research efforts in engineering photorespiration are mainly based on the modification of chloroplast glycolate metabolic steps, only limited studies have explored optimizations in other photorespiratory metabolic steps. Here, we engineered an imGS bypass within the rice mitochondria to bypass the photorespiratory glycine toward glycine betaine, thereby, improving the photosynthetic carbon fixation in rice. The imGS transgenic rice plants exhibited significant accumulation of glycine betaine, reduced photorespiration, and elevated photosynthesis and photosynthate levels. Additionally, the introduction of imGS bypass into rice leads to an increase in the number of branches and grains per panicle which may be related to cytokinin and gibberellin signaling pathways. Taken together, these results suggest diverting mitochondrial glycine from photorespiration toward glycine betaine synthesis can effectively enhance carbon fixation and panicle architecture in rice, offering a promising strategy for developing functional mitochondrial photorespiratory bypasses with the potential to enhance plant productivity.

Soybean rust (SBR), caused by an obligate biotrophic pathogen Phakopsora pachyrhizi, is a devastating disease of soybean worldwide. However, the mechanisms underlying plant invasion by P. pachyrhizi are poorly understood, which hinders the development of effective control strategies for SBR. Here we performed detailed histological characterization on the infection cycle of P. pachyrhizi in soybean and conducted a high-resolution transcriptional dissection of P. pachyrhizi during infection. This revealed P. pachyrhizi infection leads to significant changes in gene expression with 10 co-expressed gene modules, representing dramatic transcriptional shifts in metabolism and signal transduction during different stages throughout the infection cycle. Numerous genes encoding secreted protein are biphasic expressed, and are capable of inhibiting programmed cell death triggered by microbial effectors. Notably, three co-expressed P. pachyrhizi apoplastic effectors (PpAE1, PpAE2, and PpAE3) were found to suppress plant immune responses and were essential for P. pachyrhizi infection. Double-stranded RNA coupled with nanomaterials significantly inhibited SBR infection by targeting PpAE1, PpAE2, and PpAE3, and provided long-lasting protection to soybean against P. pachyrhizi. Together, this study revealed prominent changes in gene expression associated with SBR and identified P. pachyrhizi virulence effectors as promising targets of RNA interference-based soybean protection strategy against SBR.

Besides playing a crucial role in plant immunity via the nonexpressor of pathogenesis-related (NPR) proteins, increasing evidence shows that salicylic acid (SA) can also regulate plant root growth. However, the transcriptional regulatory network controlling this SA response in plant roots is still unclear. Here, we found that NPR1 and WRKY45, the central regulators of SA response in rice leaves, control only a reduced sector of the root SA signaling network. We demonstrated that SA attenuates root growth via a novel NPR1/WRKY45-independent pathway. Furthermore, using regulatory network analysis and mutant characterization, we identified a set of new NPR1/WRKY45-independent regulators that conservedly modulate the root development and root-associated microbiota composition in both Oryza sativa (monocot) and Arabidopsis thaliana (dicot) in response to SA. Our results established the SA signaling as a central element regulating plant root functions under ecologically relevant conditions. These results provide new insights to understand how regulatory networks control plant responses to abiotic and biotic stresses.

Gene innovation plays an essential role in trait evolution. Rhizobial symbioses, the most important N₂-fixing agent in agricultural systems that exists mainly in Leguminosae, is one of the most attractive evolution events. However, the gene innovations underlying Leguminosae root nodule symbiosis (RNS) remain largely unknown. Here, we investigated the gene gain event in Leguminosae RNS evolution through comprehensive phylogenomic analyses. We revealed that Leguminosae-gain genes were acquired by gene duplication and underwent a strong purifying selection. Kyoto Encyclopedia of Genes and Genomes analyses showed that the innovated genes were enriched in flavonoid biosynthesis pathways, particular downstream of chalcone synthase (CHS). Among them, Leguminosae-gain type Ⅱ chalcone isomerase (CHI) could be further divided into CHI1A and CHI1B clades, which resulted from the products of tandem duplication. Furthermore, the duplicated CHI genes exhibited exon–intron structural divergences evolved through exon/intron gain/loss and insertion/deletion. Knocking down CHI1B significantly reduced nodulation in Glycine max (soybean) and Medicago truncatula; whereas, knocking down its duplication gene CHI1A had no effect on nodulation. Therefore, Leguminosae-gain type Ⅱ CHI participated in RNS and the duplicated CHI1A and CHI1B genes exhibited RNS functional divergence. This study provides functional insights into Leguminosae-gain genetic innovation and sub-functionalization after gene duplication that contribute to the evolution and adaptation of RNS in Leguminosae.

Generation of crops with low phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP₆)) is an important breeding direction, but such plants often display less desirable agronomic traits. In this study, through ethyl methanesulfonate-mediated mutagenesis, we found that inositol 1,3,4-trisphosphate 5/6-kinase 4 (ITPK4), which is essential for producing InsP₆, is a critical regulator of salt tolerance in Arabidopsis. Loss of function of ITPK4 gene leads to reduced root elongation under salt stress, which is primarily because of decreased root meristem length and reduced meristematic cell number. The itpk4 mutation also results in increased root hair density and increased accumulation of reactive oxygen species during salt exposure. RNA sequencing assay reveals that several auxin-responsive genes are down-regulated in the itpk4-1 mutant compared to the wild-type. Consistently, the itpk4-1 mutant exhibits a reduced auxin level in the root tip and displays compromised gravity response, indicating that ITPK4 is involved in the regulation of the auxin signaling pathway. Through suppressor screening, it was found that mutation of Multidrug Resistance Protein 5 (MRP5)5 gene, which encodes an ATP-binding cassette (ABC) transporter required for transporting InsP₆ from the cytoplasm into the vacuole, fully rescues the salt hypersensitivity of the itpk4-1 mutant, but in the itpk4-1 mrp5 double mutant, InsP₆ remains at a very low level. These results imply that InsP₆ homeostasis rather than its overall amount is beneficial for stress tolerance in plants. Collectively, this study uncovers a pair of gene mutations that confer low InsP₆ content without impacting stress tolerance, which offers a new strategy for creating “low-phytate” crops.

Insects secret chemosensory proteins (CSPs) into plant cells as potential effector proteins during feeding. The molecular mechanisms underlying how CSPs activate plant immunity remain largely unknown. We show that CSPs from six distinct insect orders induce dwarfism when overexpressed in Nicotiana benthamiana. Agrobacterium-mediated transient expression of Nilaparvata lugens CSP11 (NlCSP11) triggered cell death and plant dwarfism, both of which were dependent on ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), N requirement gene 1 (NRG1) and SENESCENCE-ASSOCIATED GENE 101 (SAG101), indicating the activation of effector-triggered immunity (ETI) in N. benthamiana. Overexpression of NlCSP11 led to stronger systemic resistance against Pseudomonas syringae DC3000 lacking effector HopQ1-1 and tobacco mosaic virus, and induced higher accumulation of salicylic acid (SA) in uninfiltrated leaves compared to another effector XopQ that is recognized by a Toll-interleukin-1 receptor (TIR) domain nucleotide-binding leucine-rich repeat receptor (TNL) called ROQ1 in N. benthamiana. Consistently, NlCSP11-induced dwarfism and systemic resistance, but not cell death, were abolished in N. benthamiana transgenic line expressing the SA-degrading enzyme NahG. Through large-scale virus-induced gene silencing screening, we identified a TNL protein that mediates the recognition of CSPs (RCSP), including aphid effector MP10 that triggers resistance against aphids in N. benthamiana. Co-immunoprecipitation, bimolecular fluorescence complementation and AlphaFold2 prediction unveiled an interaction between NlCSP11 and RCSP. Interestingly, RCSP does not contain the conserved catalytic glutamic acid in the TIR domain, which is required for TNL function. Our findings point to enhanced ETI and systemic resistance by a TNL protein via hyperactivation of the SA pathway. Moreover, RCSP is the first TNL identified to recognize an insect effector.

ABSCISIC ACID-INSENSITIVE 4 (ABI4) is a pivotal transcription factor which coordinates multiple aspects of plant growth and development as well as plant responses to environmental stresses. ABI4 has been shown to be involved in regulating seedling photomorphogenesis; however, the underlying mechanism remains elusive. Here, we show that the role of ABI4 in regulating photomorphogenesis is generally regulated by sucrose, but ABI4 promotes hypocotyl elongation of Arabidopsis seedlings under blue (B) light under all tested sucrose concentrations. We further show that ABI4 physically interacts with PHYTOCHROME INTERACTING FACTOR 4 (PIF4), a well-characterized growth-promoting transcription factor, and post-translationally promotes PIF4 protein accumulation under B light. Further analyses indicate that ABI4 directly interacts with the B light photoreceptors cryptochromes (CRYs) and inhibits the interactions between CRYs and PIF4, thus relieving CRY-mediated repression of PIF4 protein accumulation. In addition, while ABI4 could directly activate its own expression, CRYs enhance, whereas PIF4 inhibits, ABI4-mediated activation of the ABI4 promoter. Together, our study demonstrates that the ABI4–PIF4 module plays an important role in mediating CRY-induced B light signaling in Arabidopsis.

Dissecting the genetic control of apple fruit harvest date (AFHD) into multiple Mendelian factors poses a significant challenge in modern genetics. Here, a quantitative trait locus (QTL) for AFHD was fine-mapped to the NAC transcription factor (TF) MdNAC18 within the interval defined by the overlap of QTLs Z03.5/Z03.6 and F03.2/F03.3. One direct target of MdNAC18 is the ethylene biosynthesis gene MdACO1. The single nucleotide polymorphisms (SNPs) SNP517 and SNP958 in the MdNAC18 coding sequence modulated activation of MdACO1 by MdNAC18. SNP1229 in the MdACO1 promoter destroyed the MdNAC18 binding site and thus abolished MdNAC18 binding. SNP517 and SNP958 also affected MdNAC18 activation of the TF gene MdARF5; MdARF5 activates the ethylene biosynthesis gene MdACS1. SNP517 and SNP958 in MdNAC18, SNP1229 and SNP769 (linked to InDel62) in MdACO1, and InDel162 in MdACS1 constituted a genetic variation network. The genetic effect of this network on AFHD was estimated as 60.3 d, accounting for 52.6% of the phenotype variation of the training population. The joint effects of these polymorphisms increased the accuracy of a genomics-assisted prediction (GAP) model for AFHD (r = 0.7125). Together, our results suggest that genetic variation in MdNAC18 affects AFHD by modulating ethylene biosynthesis and provide an optimized GAP model for apple breeding.

Poplar plantations are often established on nitrogen-poor land, and poplar growth and wood formation are constrained by low nitrogen (LN) availability. However, the molecular mechanisms by which specific genes regulate wood formation in acclimation to LN availability remain unclear. Here, we report a previously unrecognized module, basic region/leucine zipper 55 (PtobZIP55)–PtoMYB170, which regulates the wood formation of Populus tomentosa in acclimation to LN availability. PtobZIP55 was highly expressed in poplar wood and induced by LN. Altered wood anatomical properties and increased lignification were detected in PtobZIP55-overexpressing poplars, whereas the opposite results were detected in PtobZIP55-knockout poplars. Molecular and transgenic analyses revealed that PtobZIP55 directly binds to the promoter sequence of PtoMYB170 to activate its transcription. The phenotypes of PtoMYB170 transgenic poplars were similar to those of PtobZIP55 transgenic poplars under LN conditions. Further molecular analyses revealed that PtoMYB170 directly bound the promoter sequences of lignin biosynthetic genes to activate their transcription to increase lignin concentrations in LN-treated poplar wood. These results suggest that PtobZIP55 activates PtoMYB170 transcription, which in turn positively regulates lignin biosynthetic genes, increasing lignin deposition in the wood of P. tomentosa in the context of acclimation to LN availability.

Wheat culms, comprising four to six internodes, are critically involved in determining plant height and lodging resistance, essential factors for field performance and regional adaptability. This study revealed the regulatory function of miR319 in common wheat plant height. Repression of tae-miR319 through short tandem target mimics (STTM) caused an increased plant height, while overexpression (OE) of tae-miR319 had the opposite effect. Overexpressing a miR319-resistant target gene TaPCF8 (rTaPCF8), increased plant height. TaPCF8 acted as a transcription repressor of downstream genes TaIAAs, which interact physically with TaSPL14. The significant differences of indole-3-acetic acid (IAA) contents indicate the involvement of auxin pathway in miR319-mediated plant height regulation. Finally, we identified two TaPCF8 haplotypes in global wheat collections. TaPCF8-5A-Hap2, as per association and evolution examinations, was subjected to strong substantial selection throughout wheat breeding. This haplotype, associated with shorter plant height, aligns with global breeding requirements. Consequently, in high-yield wheat breeding, we proposed a potential molecular marker for marker-assisted selection (MAS). Our findings offer fresh perspectives into the molecular mechanisms that underlie the miR319–TaPCF8 module's regulation of plant height by orchestrating auxin signaling and biosynthesis in wheat.

Light is one of the most essential environmental factors that tightly and precisely control various physiological and developmental processes in plants. B-box CONTAINING PROTEINs (BBXs) play central roles in the regulation of light-dependent development. In this study, we report that BBX9 is a positive regulator of light signaling. BBX9 interacts with the red light photoreceptor PHYTOCHROME B (phyB) and transcription factors PHYTOCHROME-INTERACTING FACTORs (PIFs). phyB promotes the stabilization of BBX9 in light, while BBX9 inhibits the transcriptional activation activity of PIFs. In turn, PIFs directly bind to the promoter of BBX9 to repress its transcription. On the other hand, BBX9 associates with the positive regulator of light signaling, BBX21, and enhances its biochemical activity. BBX21 associates with the promoter regions of BBX9 and transcriptionally up-regulates its expression. Collectively, this study unveiled that BBX9 forms a negative feedback loop with PIFs and a positive one with BBX21 to ensure that plants adapt to fluctuating light conditions.

The calcineurin B-like protein (CBL)-CBL-interacting protein kinase (CIPK) Ca²⁺ sensors play crucial roles in the plant's response to drought stress. However, there have been few reports on the synergistic regulation of drought stress by CBL-CIPK and abscisic acid (ABA) core signaling components. In this study, we discovered that ZmCIPK33 positively regulates drought resistance in maize. ZmCIPK33 physically interacts with and is enhanced by phosphorylation from ZmSnRK2.10. Drought stress can activate ZmCIPK33, which is partially dependent on ZmSnRK2.10. ZmCIPK33 in combination with ZmSnRK2.10 can activate the slow anion channel ZmSLAC1 in Xenopus laevis oocytes independently of CBLs, whereas ZmCIPK33 or ZmSnRK2.10 alone is unable to do so. Furthermore, ZmCIPK33 phosphorylates ZmPP2C11 at Ser60, which leads to a reduction in the interaction between ZmPP2C11 and ZmEAR1 (the ortholog of Arabidopsis Enhancer of ABA co-Receptor 1) and weakens the phosphatase activity of ZmPP2C11, consequently, enhancing the activity of ZmSnRK2.10 in an in vitro assay and in the in-gel assay of the zmcipk33 mutant. Our findings provide novel insights into the molecular mechanisms underlying the reciprocal enhancement of Ca²⁺ and ABA signaling under drought stress in maize.

Wild species of domesticated crops provide valuable genetic resources for resistance breeding. Prunus davidiana, a wild relative of peach with high heterozygosity and diverse stress tolerance, exhibits high resistance against aphids. However, the highly heterozygous genome of P. davidiana makes determining the underlying factors influencing resistance traits challenging. Here, we present the 501.7 Mb haplotype-resolved genome assembly of P. davidiana. Genomic comparisons of the two haplotypes revealed 18,152 structural variations, 2,699 Pda_hap1-specific and 2,702 Pda_hap2-specific genes, and 1,118 allele-specific expressed genes. Genome composition indicated 4.1% of the P. davidiana genome was non-peach origin, out of which 94.5% was derived from almond. Based on the haplotype genome, the aphid resistance quantitative trait locus (QTL) was mapped at the end of Pda03. From the aphid resistance QTL, PdaWRKY4 was identified as the major dominant gene, with a 9-bp deletion in its promoter of the resistant phenotype. Specifically, PdaWRKY4 regulates aphid resistance by promoting PdaCYP716A1-mediated anti-aphid metabolite betulin biosynthesis. Moreover, we employed a genome design to develop a breeding workflow for rapidly and precisely producing aphid-resistant peaches. In conclusion, this study identifies a novel aphid resistance gene and provides insights into genome design for the development of resistant fruit cultivars.