Myosin XI-mediated BIK1 recruitment to nanodomains facilitates FLS2-BIK1 complex formation during innate immunity in Arabidopsis.

Plants rely on immune receptor complexes at the cell surface to perceive microbial molecules and transduce these signals into the cell to regulate immunity. Various immune receptors and associated proteins are often dynamically distributed in specific nanodomains on the plasma membrane (PM). However, the exact molecular mechanism and functional relevance of this nanodomain targeting in plant immunity regulation remain largely unknown. By utilizing high spatiotemporal resolution imaging and single-particle tracking analysis, we show that myosin XIK interacts with remorin to recruit and stabilize PM-associated kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) within immune receptor FLAGELLIN SENSING 2 (FLS2)-containing nanodomains. This recruitment facilitates FLS2/BIK1 complex formation, leading to the full activation of BIK1-dependent defense responses upon ligand perception. Collectively, our findings provide compelling evidence that myosin XI functions as a molecular scaffold to enable a spatially confined complex assembly within nanodomains. This ensures the presence of a sufficient quantity of preformed immune receptor complex for efficient signaling transduction from the cell surface.

Heterotrimeric Gα-subunit regulates flower and fruit development in CLAVATA signaling pathway in cucumber.

Flowers and fruits are the reproductive organs in plants and play essential roles in natural beauty and the human diet. CLAVATA (CLV) signaling has been well characterized as regulating floral organ development by modulating shoot apical meristem (SAM) size; however, the signaling molecules downstream of the CLV pathway remain largely unknown in crops. Here, we found that functional disruption of CsCLV3 peptide and its receptor CsCLV1 both resulted in flowers with extra organs and stumpy fruits in cucumber. A heterotrimeric G protein α-subunit (CsGPA1) was shown to interact with CsCLV1. Csgpa1 mutant plants derived from gene editing displayed significantly increased floral organ numbers and shorter and wider fruits, a phenotype resembling that of Csclv mutants in cucumber. Moreover, the SAM size was enlarged and the longitudinal cell size of fruit was decreased in Csgpa1 mutants. The expression of the classical stem cell regulator WUSCHEL (WUS) was elevated in the SAM, while the expression of the fruit length stimulator CRABS CLAW (CRC) was reduced in the fruit of Csgpa1 mutants. Therefore, the Gα-subunit CsGPA1 protein interacts with CsCLV1 to inhibit floral organ numbers but promote fruit elongation, via repressing CsWUS expression and activating CsCRC transcription in cucumber. Our findings identified a new player in the CLV signaling pathway during flower and fruit development in dicots, increasing the number of target genes for precise manipulation of fruit shape during crop breeding.

A comprehensive review of aptamer screening and application for lateral flow strip: Current status and future perspectives.

The detection of biomarkers is of great significance for medical diagnosis, food safety, environmental monitoring, and agriculture. However, bio-detection technology at present often necessitates complex instruments, expensive reagents, specialized expertise, and prolonged procedures, making it challenging to fulfill the demand for rapid, sensitive, user-friendly, and economical testing. In contrast, lateral flow strip (LFS) technology offers simple, fast, and visually accessible detection modality, allowing real-time analysis of clinical specimens, thus finding widespread utility across various domains. Within the realm of LFS, the application of aptamers as molecular recognition probes presents distinct advantages over antibodies, including cost-effectiveness, smaller size, ease of synthesis, and chemical stability. In recent years, aptamer-based LFS has found extensive application in qualitative, semi-quantitative, and quantitative detection across food safety, environmental surveillance, clinical diagnostics, and other domains. This review provided a concise overview of different aptamer screening methodologies, selection strategies, underlying principles, and procedural, elucidating their respective advantages, limitations, and applications. Additionally, we summarized recent strategies and mechanisms for aptamer-based LFS, such as the sandwich and competitive methods. Furthermore, we classified LFSs constructed based on aptamers, considering the rapid advancements in this area, and discussed their applications in biological and chemical detection. Finally, we delved into the current challenges and future directions in the development of aptamer and aptamer-based LFS. Although this review was not thoroughly, it would serve as a valuable reference for understanding the research progress of aptamer-based LFS and aid in the development of new types of aptasensors.

The AGAMOUS-LIKE 16-GENERAL REGULATORY FACTOR 1 module regulates axillary bud outgrowth via catabolism of abscisic acid in cucumber.

Lateral branches are important components of shoot architecture and directly affect crop yield and production cost. Although sporadic studies have implicated abscisic acid (ABA) biosynthesis in axillary bud outgrowth, the function of ABA catabolism and its upstream regulators in shoot branching remain elusive. Here, we showed that the MADS-box transcription factor AGAMOUS-LIKE 16 (CsAGL16) is a positive regulator of axillary bud outgrowth in cucumber (Cucumis sativus). Functional disruption of CsAGL16 led to reduced bud outgrowth, whereas overexpression of CsAGL16 resulted in enhanced branching. CsAGL16 directly binds to the promoter of the ABA 8'-hydroxylase gene CsCYP707A4 and promotes its expression. Loss of CsCYP707A4 function inhibited axillary bud outgrowth and increased ABA levels. Elevated expression of CsCYP707A4 or treatment with an ABA biosynthesis inhibitor largely rescued the Csagl16 mutant phenotype. Moreover, cucumber General Regulatory Factor 1 (CsGRF1) interacts with CsAGL16 and antagonizes CsAGL16-mediated CsCYP707A4 activation. Disruption of CsGRF1 resulted in elongated branches and decreased ABA levels in the axillary buds. The Csagl16 Csgrf1 double mutant exhibited a branching phenotype resembling that of the Csagl16 single mutant. Therefore, our data suggest that the CsAGL16-CsGRF1 module regulates axillary bud outgrowth via CsCYP707A4-mediated ABA catabolism in cucumber. Our findings provide a strategy to manipulate ABA levels in axillary buds during crop breeding to produce desirable branching phenotypes.

CsRAXs negatively regulate leaf size and fruiting ability through auxin glycosylation in cucumber.

Leaves are the main photosynthesis organ that directly determines crop yield and biomass. Dissecting the regulatory mechanism of leaf development is crucial for food security and ecosystem turn-over. Here, we identified the novel function of R2R3-MYB transcription factors CsRAXs in regulating cucumber leaf size and fruiting ability. Csrax5 single mutant exhibited enlarged leaf size and stem diameter, and Csrax1/2/5 triple mutant displayed further enlargement phenotype. Overexpression of CsRAX1 or CsRAX5 gave rise to smaller leaf and thinner stem. The fruiting ability of Csrax1/2/5 plants was significantly enhanced, while that of CsRAX5 overexpression lines was greatly weakened. Similarly, cell number and free auxin level were elevated in mutant plants while decreased in overexpression lines. Biochemical data indicated that CsRAX1/5 directly promoted the expression of auxin glucosyltransferase gene CsUGT74E2. Therefore, our data suggested that CsRAXs function as repressors for leaf size development by promoting auxin glycosylation to decrease free auxin level and cell division in cucumber. Our findings provide new gene targets for cucumber breeding with increased leaf size and crop yield.

The exocyst subunit CsExo70B promotes both fruit length and disease resistance via regulating receptor kinase abundance at plasma membrane in cucumber.

Plant defence against pathogens generally occurs at the expense of growth and yield. Uncoupling the inverse relationship between growth and defence is of great importance for crop breeding, while the underlying genes and regulatory mechanisms remain largely elusive. The exocytosis complex was shown to play an important role in the trafficking of receptor kinases (RKs) to the plasma membrane (PM). Here, we found a Cucumis sativus exocytosis subunit Exo70B (CsExo70B) regulates the abundance of both development and defence RKs at the PM to promote fruit elongation and disease resistance in cucumber. Knockout of CsExo70B resulted in shorter fruit and susceptibility to pathogens. Mechanistically, CsExo70B associates with the developmental RK CsERECTA, which promotes fruit longitudinal growth in cucumber, and contributes to its accumulation at the PM. On the other side, CsExo70B confers to the spectrum resistance to pathogens in cucumber via a similar regulatory module of defence RKs. Moreover, CsExo70B overexpression lines showed an increased fruit yield as well as disease resistance. Collectively, our work reveals a regulatory mechanism that CsExo70B promotes both fruit elongation and disease resistance by maintaining appropriate RK levels at the PM and thus provides a possible strategy for superior cucumber breeding with high yield and robust pathogen resistance.

The WRKY17-WRKY50 complex modulates anthocyanin biosynthesis to improve drought tolerance in apple.

Drought stress is increasing worldwide due to global warming, which severely reduces apple (Malus domestica) yield. Clarifying the basis of drought tolerance in apple could accelerate the molecular breeding of drought-tolerant cultivars to maintain apple production. We identified a transcription factor MdWRKY50 by yeast two-hybrid (Y2H) assays as an interactor of the drought-tolerant protein MdWRKY17, and confirmed their interaction by bimolecular fluorescence complementation (BiFC) and pull-down assays. MdWRKY50 was induced by drought and when overexpressed in apple, conferred transgenic apple plants enhanced drought tolerance by directly binding to the promoter of anthocyanin synthetic gene Chalcone synthase (MdCHS) to upregulate its expression for higher anthocyanin. Increased anthocyanin relieves apple plants from oxidative damage under drought stress. MdWRKY50 RNA-interference transgenic apple plants showed opposite phenotypes. The dimerization of MdWRKY50 with mutated MdWRKY17DP mimicking drought-induced phosphorylation by the mitogen-activated protein kinase kinase 2 (MEK2)-MPK6 cascade, compared with MdWRKY17AP and MdWRKY17, further promoted anthocyanin biosynthesis, suggesting dimerization with MdWRKY17 makes MdWRKY50 more powerful in promoting anthocyanin biosynthesis under drought stress. Taken together, we isolated an entire MEK2-MAPK6-MdWRKY17-MdWRKY50-MdCHS pathway for drought tolerance and generated transgenic apple germplasm with enhanced drought tolerance and higher anthocyanin levels.

Genome-Wide Analysis of Exocyst Complex Subunit Exo70 Gene Family in Cucumber.

Cucumber (Cucumis sativus L.) is an important vegetable worldwide, but its yield is affected by a wide range of pathogens and pests. As the major subunit of the exocyst complex, the roles of Exo70 members have been shown in Arabidopsis and rice, but their function are unknown in cucumber. Here, we identified 18 CsExo70 members in cucumber, which were divided into three groups (Exo70.1-Exo70.3) and nine subgroups (Exo70A-Exo70I) based on the phylogenetic tree. Subsequently, systematical analyses were performed, including collinearity, gene structure, cis-acting elements, conserved motifs, expression patterns, and subcellular localization. Our results showed that CsExo70 genes were generally expressed in all tissues, and CsExo70C1 and CsExo70C2 were highly expressed in the stamen. Moreover, the expression levels of most CsExo70 genes were induced by Pseudomonas syringae pv. lachrymans (Psl) and Fusarium oxysporum f. sp. cucumerinum Owen (Foc), especially CsExo70E2 and CsExo70H3. In addition, these CsExo70s displayed similar location patterns with discrete and punctate signals in the cytoplasm. Together, our results indicate that CsExo70 members may be involved in plant development and resistance, and provide a reference for future in-depth studies of Exo70 genes in cucumber.

Pollen tube emergence is mediated by ovary-expressed ALCATRAZ in cucumber.

Pollen tube guidance within female tissues of flowering plants can be divided into preovular guidance, ovular guidance and a connecting stage called pollen tube emergence. As yet, no female factor has been identified to positively regulate this transition process. In this study, we show that an ovary-expressed bHLH transcription factor Cucumis sativus ALCATRAZ (CsALC) functions in pollen tube emergence in cucumber. CsALC knockout mutants showed diminished pollen tube emergence, extremely reduced entry into ovules, and a 95% reduction in female fertility. Further examination showed two rapid alkalinization factors CsRALF4 and CsRALF19 were less expressed in Csalc ovaries compared to WT. Besides the loss of male fertility derived from precocious pollen tube rupture as in Arabidopsis, Csralf4 Csralf19 double mutants exhibited a 60% decrease in female fertility due to reduced pollen tube distribution and decreased ovule targeting efficiency. In brief, CsALC regulates female fertility and promotes CsRALF4/19 expression in the ovary during pollen tube guidance in cucumber.

Natural variation in CRABS CLAW contributes to fruit length divergence in cucumber.

Fruit length is a key domestication trait that affects crop yield and appearance. Cucumber (Cucumis sativus) fruits vary from 5 to 60 cm in length. Despite the identification of several regulators and multiple quantitative trait loci (QTLs) underlying fruit length, the natural variation, and molecular mechanisms underlying differences in fruit length are poorly understood. Through map-based cloning, we identified a nonsynonymous polymorphism (G to A) in CRABS CLAW (CsCRC) as underlying the major-effect fruit size/shape QTL FS5.2 in cucumber. The short-fruit allele CsCRCA is a rare allele that has only been found in round-fruited semi-wild Xishuangbanna cucumbers. A near-isogenic line (NIL) homozygous for CsCRCA exhibited a 34∼39% reduction in fruit length. Introducing CsCRCG into this NIL rescued the short-fruit phenotype, and knockdown of CsCRCG resulted in shorter fruit and smaller cells. In natural cucumber populations, CsCRCG expression was positively correlated with fruit length. Further, CsCRCG, but not CsCRCA, targets the downstream auxin-responsive protein gene CsARP1 to regulate its expression. Knockout of CsARP1 produced shorter fruit with smaller cells. Hence, our work suggests that CsCRCG positively regulates fruit elongation through transcriptional activation of CsARP1 and thus enhances cell expansion. Using different CsCRC alleles provides a strategy to manipulate fruit length in cucumber breeding.

A surface-receptor-coupled G protein regulates plant immunity through nuclear protein kinases.

Plants employ cell-surface-localized pattern recognition receptors (PRRs) to recognize immunogenic patterns and activate defenses. How these receptors regulate immune signaling in the nucleus is not well understood. Our previous studies showed that BIK1, a central kinase associated with PRRs, phosphorylates a plant-specific Gα protein called extra-large G protein 2 (XLG2) to positively regulate immunity. Here, we show that this phosphorylation promotes XLG2 nuclear translocation, which is essential for antibacterial immunity. XLG2 interacts with nuclear-localized MUT9-like kinases (MLKs) to regulate transcriptome programming. MLKs negatively regulate plant immunity in a kinase activity-dependent manner, whereas XLG2 promotes defense gene expression and antibacterial immunity likely by inhibiting MLK kinase activity. A C-terminal motif in MLKs is essential for the interaction with XLG2, and this motif is required for the XLG2-mediated defense activation. Together, our findings reveal a previously unknown pathway and mechanisms by which cell surface receptors regulate transcriptome during pathogen invasion.

The CsHEC1-CsOVATE module contributes to fruit neck length variation via modulating auxin biosynthesis in cucumber.

Fruit neck is the proximal portion of the fruit with undesirable taste that has detrimental effects on fruit shape and commercial value in cucumber. Despite the dramatic variations in fruit neck length of cucumber germplasms, the genes and regulatory mechanisms underlying fruit neck elongation remain mysterious. In this study, we found that Cucumis sativus HECATE1 (CsHEC1) was highly expressed in fruit neck. Knockout of CsHEC1 resulted in shortened fruit neck and decreased auxin accumulation, whereas overexpression of CsHEC1 displayed the opposite effects, suggesting that CsHEC1 positively regulated fruit neck length by modulating local auxin level. Further analysis showed that CsHEC1 directly bound to the promoter of the auxin biosynthesis gene YUCCA4 (CsYUC4) and activated its expression. Enhanced expression of CsYUC4 resulted in elongated fruit neck and elevated auxin content. Moreover, knockout of CsOVATE resulted in longer fruit neck and higher auxin. Genetic and biochemical data showed that CsOVATE physically interacted with CsHEC1 to antagonize its function by attenuating the CsHEC1-mediated CsYUC4 transcriptional activation. In cucumber germplasms, the expression of CsHEC1 and CsYUC4 positively correlated with fruit neck length, while that of CsOVATE showed a negative correlation. Together, our results revealed a CsHEC1-CsOVATE regulatory module that confers fruit neck length variation via CsYUC4-mediated auxin biosynthesis in cucumber.

Molecular basis of high temperature-induced bolting in lettuce revealed by multi-omics analysis.

BACKGROUND: High temperature induces early bolting in lettuce (Lactuca sativa L.), which affects both quality and production. However, the molecular mechanism underlying high temperature-induced bolting is still limited. RESULTS: We performed systematical analysis of morphology, transcriptome, miRNAs and methylome in lettuce under high temperature treatment. Through a comparison of RNA-Seq data between the control and the high temperature treated lettuces at different time points totally identified 2944 up-regulated genes and 2203 down-regulated genes, which cover three floral pathways including photoperiod, age and gibberellin (GA) pathways. Genome wide analysis of miRNAs and methylome during high temperature treatment indicated miRNAs and DNA methylation might play a role controlling gene expression during high temperature-induced bolting. miRNA targets included some protein kinase family proteins, which potentially play crucial roles in this process. CONCLUSIONS: Together, our results propose a possible regulation network involved in high temperature-induced bolting.

Two plant NLR proteins confer strain-specific resistance conditioned by an effector from Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae (Psa) causes bacterial canker, a devastating disease threatening the Actinidia fruit industry. In a search for non-host resistance genes against Psa, we find that the nucleotide-binding leucine-rich repeat receptor (NLR) protein ZAR1 from both Arabidopsis and Nicotiana benthamiana (Nb) recognizes HopZ5 and triggers cell death. The recognition requires ZED1 in Arabidopsis and JIM2 in Nb plants, which are members of the ZRK pseudokinases and known components of the ZAR1 resistosome. Surprisingly, Arabidopsis ZAR1 and RPM1, another NLR known to recognize HopZ5, confer disease resistance to HopZ5 in a strain-specific manner. Thus, ZAR1, but not RPM1, is solely required for resistance to P. s. maculicola ES4326 (Psm) carrying hopZ5, whereas RPM1 is primarily required for resistance to P. s. tomato DC3000 (Pst) carrying hopZ5. Furthermore, the ZAR1-mediated resistance to Psm hopZ5 in Arabidopsis is insensitive to SOBER1, which encodes a deacetylase known to suppress the RPM1-mediated resistance to Pst hopZ5. In addition, hopZ5 enhances P. syringae virulence in the absence of ZAR1 or RPM1 and that SOBER1 abolishes such virulence function. Together the study suggests that ZAR1 may be used for improving Psa resistance in Actinidia and uncovers previously unknown complexity of effector-triggered immunity and effector-triggered virulence.

SPATULA and ALCATRAZ confer female sterility and fruit cavity via mediating pistil development in cucumber.

Fruits and seeds play essential roles in plant sexual reproduction and the human diet. Successful fertilization involves delivery of sperm in the pollen tube to the egg cell within the ovary along the transmitting tract (TT). Fruit cavity is an undesirable trait directly affecting cucumber (Cucumis sativus) commercial value. However, the regulatory genes underlying fruit cavity formation and female fertility determination remain unknown in crops. Here, we characterized a basic Helix-Loop-Helix (bHLH) gene C. sativus SPATULA (CsSPT) and its redundant and divergent function with ALCATRAZ (CsALC) in cucumber. CsSPT transcripts were enriched in reproductive organs. Mutation of CsSPT resulted in 60% reduction in female fertility, with seed produced only in the upper portion of fruits. Csspt Csalc mutants displayed complete loss of female fertility and fruit cavity due to carpel separation. Further examination showed that stigmas in the double mutant turned outward with defective papillae identity, and extracellular matrix contents in the abnormal TT were dramatically reduced, which resulted in no path for pollen tube extension and no ovules fertilized. Biochemical and transcriptome analysis showed that CsSPT and CsALC act in homodimers and heterodimers to confer fruit cavity and female sterility by mediating genes involved in TT development, auxin-mediated signaling, and cell wall organization in cucumber.

Dual fluorescent aptasensor for simultanous and quantitative detection of sulfadimethoxine and oxytetracycin residues in animal-derived foods tissues based on mesoporous silica.

Herein, we developed a dual fluorescent aptasensor based on mesoporous silica to simultaneously detect sulfadimethoxine (SDM) and oxytetracycline (OTC) in animal-derived foods. We immobilized two types of aptamers modified with FAM and CY5 on the silica surface by base complementary pairing reaction with the cDNA modified with a carboxyl group and finally formed the aptasensor detection platform. Under optimal conditions, the detection range of the aptasensor for SDM and OTC was 3-150 ng/mL (R 2 = 0.9831) and 5-220 ng/mL (R 2 = 0.9884), respectively. The limits of detection for SDM and OTC were 2.2 and 1.23 ng/mL, respectively. The limits of quantification for SDM and OTC were 7.3 and 4.1 ng/mL, respectively. Additionally, the aptasensor was used to analyze spiked samples. The average recovery rates ranged from 91.75 to 114.65% for SDM and 89.66 to 108.94% for OTC, and all coefficients of variation were below 15%. Finally, the performance and practicability of our aptasensor were confirmed by HPLC, demonstrating good consistency. In summary, this study was the first to use the mesoporous silica-mediated fluorescence aptasensor for simultaneous detection of SDM and OTC, offering a new possibility to analyze other antibiotics, biotoxins, and biomolecules.

MPK3- and MPK6-mediated VLN3 phosphorylation regulates actin dynamics during stomatal immunity in Arabidopsis.

Upon perception of pathogens, plants can rapidly close their stomata to restrict pathogen entry into internal tissue, leading to stomatal immunity as one aspect of innate immune responses. The actin cytoskeleton is required for plant defense against microbial invaders. However, the precise functions of host actin during plant immunity remain largely unknown. Here, we report that Arabidopsis villin3 (VLN3) is critical for plant resistance to bacteria by regulating stomatal immunity. Our in vitro and in vivo phosphorylation assays show that VLN3 is a physiological substrate of two pathogen-responsive mitogen-activated protein kinases, MPK3/6. Quantitative analyses of actin dynamics and genetic studies reveal that VLN3 phosphorylation by MPK3/6 modulates actin remodeling to activate stomatal defense in Arabidopsis.

HECATE2 acts with GLABROUS3 and Tu to boost cytokinin biosynthesis and regulate cucumber fruit wart formation.

Warty fruit in cucumber (Cucumis sativus L.) is an important quality trait that greatly affects fruit appearance and market value. The cucumber wart consists of fruit trichomes (spines) and underlying tubercules, in which the existence of spines is prerequisite for tubercule formation. Although several regulators have been reported to mediate spine or tubercule formation, the direct link between spine and tubercule development remains unknown. Here, we found that the basic Helix-Loop-Helix (bHLH) gene HECATE2 (CsHEC2) was highly expressed in cucumber fruit peels including spines and tubercules. Knockout of CsHEC2 by the CRISPR/Cas9 system resulted in reduced wart density and decreased cytokinin (CTK) accumulation in the fruit peel, whereas overexpression of CsHEC2 led to elevated wart density and CTK level. CsHEC2 is directly bound to the promoter of the CTK hydroxylase-like1 gene (CsCHL1) that catalyzes CTK biosynthesis, and activated CsCHL1 expression. Moreover, CsHEC2 physically interacted with GLABROUS3 (CsGL3, a key spine regulator) and Tuberculate fruit (CsTu, a core tubercule formation factor), and such interactions further enhanced CsHEC2-mediated CsCHL1 expression. These data suggested that CsHEC2 promotes wart formation by acting as an important cofactor for CsGL3 and CsTu to directly stimulate CTK biosynthesis in cucumber. Thus, CsHEC2 can serve as a valuable target for molecular breeding of cucumber varieties with different wart density requirements.