sRNAanno-a database repository of uniformly annotated small RNAs in plants

Small RNAs(sRNAs)are essential regulatory molecules,and there are three major sRNA classes in plants:microRNAs(miRNAs),phased small interfering RNAs(phased siRNAs or phasiRNAs),and heterochromatic siRNAs(hc-siRNAs).Excluding miRNAs,the other two classes are not well annotated or available in public databases for most sequenced plant genomes.We performed a comprehensive sRNA annotation of 143 plant species that have fully sequenced genomes and next-generation sequencing sRNA data publicly available.The results are available via an online repository called sRNAanno(www.plantsRNAs.org).Compared with other public plant sRNA databases,we obtained was much more miRNA annotations,which are more complete and reliable because of the consistent and highly stringent criteria used in our miRNA annotations.sRNAanno also provides free access to genomic information for>22,721 PHAS loci and>22 million hc-siRNA loci annotated from these 143 plant species.Both miRNA and PHAS loci can be easily browsed to view their main features,and a collection of archetypal trans-acting siRNA 3(TAS3)genes were annotated separately for quick access.To facilitate the ease of sRNA annotation,sRNAanno provides free service for sRNA annotations to the community.In summary,the sRNAanno database is a great resource to facilitate genomic and genetic research on plant small RNAs.

Application of Digital PCR in the Analysis of Transgenic Soybean Plants

Detection and quantification of transgenes are important in analyzing genetically modified organisms (GMOs). Quantitative polymerase chain reaction (qPCR) is commonly utilized for such purposes. However, qPCR has certain limitations in detecting and quantifying transgenes in GMOs, such as the need of certified reference materials, a standard curve, and possible affection by inhibitors. Therefore, alternative and possibly better methods are needed. Recent advances in digital PCR technologies have promised to allow accurate quantification of nucleic acids and therefore provided another useful technique to analyze GMOs. Thermo Fisher ScientificTM has recently commercialized the Applied BiosystemsTM QuantStudioTM 3D digital PCR system that can be used for a wide range of applications involving nucleic acids. It will be beneficial to the scientific community to show the applicability of this digital PCR system in detecting and quantifying transgenes in GMOs. In the present study, the transgenes present in the Roundup Ready Soybean (RR1, event 40-3-2) and Roundup Ready Soybean 2 (RR2, event MON89788) developed by Monsanto Corporation were analyzed by using this digital PCR system. The qPCR analysis results were included for comparison. Using specifically designed TaqMan assays, as low as 1% of the RR1 or RR2 soybean material was reliably detected and quantified on the dPCR platform. Therefore, digital PCR is a sensitive and reliable method to analyze the RR transgenic soybeans, and should be another useful tool for analyzing other transgenic plants.

Direct Organogenesis from Cotyledonary Node Explants of Cucurbita pepo (L.)—An Important Zucchini Type Vegetable Crop

Cotyledonary node explants from 3 - 5-d-old seedling derived explants of Cucurbita pepo (L.) produced multiple shoots in Murashige and Skoog (MS) salts B5 vitamins containing medium in the presence of N6-benzylamino-purine BAP 1 mg/L + Thidiazuron TDZ 0.05 mg/L. After 4 weeks explants were subcultured to medium containing MS salts B5 Vitamins BAP 0.5 mg/L, Gibberellic acid GA3 1 mg/L and L-Glutamine 15 mg/L. Periodic excision of regenerated shoots from the explants increased shoot efficiency during subculture. The combination of L-Glutamine with BA and GA3 significantly increased the shoot proliferation. Elongated shoots were excised from shoot clumps and transferred to rooting medium containing indole-3-butyric acid (IBA, 1.0 mg/L). The rooted plants were hardened in small pot containing standardized garden soil, well developed plant transferred to greenhouse and survival rate was 85%.

Applying plant-based irrigation scheduling to assess water use efficiency of cotton following a high-biomass rye cover crop

Background:This study addressed the potential of combining a high biomass rye winter cover crop with predawn leaf water potential(ΨPD)irrigation thresholds to increase agricultural water use efficiency(WUE)in cotton.To this end,a study was conducted near Tifton,Georgia under a manually-controlled,variable-rate lateral irrigation system using a Scholander pressure chamber approach to measure leaf water potential and impose varying irrigation scheduling treatments during the growing season.ΨPDthresholds were-0.4 MPa(T1),-0.5 MPa(T2),and-0.7 MPa(T3).A winter rye cover crop or conventional tillage were utilized for T1-T3 as well.Results:Reductions in irrigation of up to 10%were noted in this study for the driest threshold(-0.7 MPa)with no reduction in lint yield relative to the-0.4 MPa and-0.5 MPa thresholds.Drier conditions during flowering(2014)limited plant growth and node production,hastened cutout,and decreased yield and WUE relative to 2015.Conclusions:We conclude thatΨPDirrigation thresholds between-0.5 MPa and-0.7 MPa appear to be viable for use in aΨPDscheduling system with adequate yield and WUE for cotton production in the southeastern U.S.Rye cover positively impacted water potential at certain points throughout the growing season but not yield or WUE indicating the potential for rye cover crops to improve water use efficiency should be tested under longer-term production scenarios.

The Relative Contribution of Non-Foliar Organs of Cotton to Yield and Related Physiological Characteristics Under Water Defi cit

Water deficit is one of the most important causes of decreased yield in cultivated plants. Non-foliar green organs in cotton play an important role in yield formation at the late growth stage. Although better photosynthetic performance was observed in a non-foliar organ （bract） compared with leaves under water deficit. However, the physiological response of each organ in cotton to water deficit has not been comprehensively studied in relation to the water status and photosynthesis characteristics. We studied the maintenance of water status of each organ in cotton by measuring their relative water content, proline content and stomatal characteristics. Water deficit significantly decreased the surface area of each organ, but to a lesser extent in non-foliar organs. Our results showed that the relative contribution of biomass accumulation of non-foliar organs increased under water deficit. Non-foliar organs （bracts and capsule wall） showed less ontogenetic decrease in O2 evolution capacity and in RuBPC activity （per dry weight） as well as better antioxidant systems than leaves at various days after anthesis. We conclude that the photosynthesis from non-foliar organs is important for increasing cotton yield especially under water deficit conditions.

Development of a panel of unigene-derived polymorphic EST–SSR markers in lentil using public database information

Lentil(Lens culinaris Medik.), a diploid(2n = 14) with a genome size greater than 4000 Mbp, is an important cool season food legume grown worldwide. The availability of genomic resources is limited in this crop species. The objective of this study was to develop polymorphic markers in lentil using publicly available curated expressed sequence tag information(ESTs). In this study, 9513 ESTs were downloaded from the National Center for Biotechnology Information(NCBI) database to develop unigene-based simple sequence repeat(SSR) markers. The ESTs were assembled into 4053 unigenes and then analyzed to identify 374 SSRs using the MISA microsatellite identification tool. Among the 374 SSRs, 26 compound SSRs were observed.Primer pairs for these SSRs were designed using Primer3 version 1.14. To classify the functional annotation of ESTs and EST–SSRs, BLASTx searches(using E-value 1 × 10-5) against the public UniP rot(http://www.uniprot.org/) and NCBI(http://www.ncbi.nlh.nih.gov/) databases were performed. Further functional annotation was performed using PLAZA(version3.0) comparative genomics and GO annotation was summarized using the Plant GO slim category. Among the synthesized 312 primers, 219 successfully amplified Lens DNA. A diverse panel of 24 Lens genotypes was used to identify polymorphic markers. A polymorphic set of 57 markers successfully discriminated the test genotypes. This set of polymorphic markers with functional annotation data could be used as molecular tools in lentil breeding.

Effect of Flood Stress on Soybean Seed Germination in the Field

Flooding is an abiotic stress that impacts soybean [Glycine max (L.) Merr.] growth and reduces seed germination. Effect of flooding on soybean plant grown at different growth stage has been previously conducted and reported. However, soybean seed germination responses to flood stress are largely unknown. The objective of this study was to elucidate flooding influence on soybean seed germination after planting in the field. The research showed that seed germination rate (SGR) of each genotype, without flood stress, was significantly different and ranged between 64.7% to 84.0% and 69.0% to 90.7% while using untreated and fungicide-treated seed (P < 0.0001), respectively. Results indicated that fungicide treatment improved soybean seed survival and germination in the field. The average of SGR of high-yielding soybean group was significantly higher than those of non-high-yielding soybean (P < 0.0001). The results indicated that high-yielding trait of each genotype was correlated with seed germination and survival. Under flood stress in the field, SGR means of untreated and fungicide-treated seed significantly decreased over eight flooding treatment times (P Flooding effect on germination between untreated and fungicide-treated seed was not significantly different (P = 0.1559). Furthermore, comparing the high-yielding and flood-tolerant soybean groups showed no difference in their SGR means over eight flooding treatment times (P = 0.7687 and P = 0.8490), indicating that soybean seed germination did not depend on genotype, yield, and flood tolerance trait, and seed treated by fungicide did not increase its germination in the field under the flood stress. Hence, it is necessary to develop new soybean seed pelleting to improve seed germination in the field under flooding conditions.

DNA methylation-mediated expression of zinc finger protein 615 affects embryonic development in Bombyx mori

Cell division and differentiation after egg fertilization are critical steps in the development of embryos from single cells to multicellular individuals and are regulated by DNA methylation via its effects on gene expression.However,the mechanisms by which DNA methylation regulates these processes in insects remain unclear.Here,we studied the impacts of DNA methylation on early embryonic development in Bombyx mori.Genome methylation and transcriptome analysis of early embryos showed that DNA methylation events mainly occurred in the 5'region of protein metabolism-related genes.The transcription factor gene zinc finger protein 615(ZnF615)was methylated by DNA methyltransferase 1(Dnmt1)to be up-regulated and bind to protein metabolism-related genes.Dnmt1 RNA interference(RNAi)revealed that DNA methylation mainly regulated the expression of nonmethylated nutrient metabolism-related genes through ZnF615.The same sites in the ZnF615 gene were methylated in ovaries and embryos.Knockout of ZnF615 using CRISPR/Cas9 gene editing decreased the hatching rate and egg number to levels similar to that of Dnmt1 knockout.Analysis of the ZnF615 methylation rate revealed that the DNA methylation pattern in the parent ovary was maintained and doubled in the offspring embryo.Thus,Dnmt1-mediated intragenic DNA methylation of the transcription factor ZnF615 enhances its expression to ensure ovarian and embryonic development.

Testing allometric scaling relationships in plant roots

Background:Metabolic scaling theory predicts that plant productivity and biomass are both size-dependent.However,this theory has not yet been tested in plant roots.Methods:In this study,we tested how metabolic scaling occurs in plants using a comprehensive plant root dataset made up of 1016 observations from natural habitats.We generated metabolic scaling exponents by logtransformation of root productivity versus biomass.Results:Results showed that the metabolic scaling exponents of fine root(<2mm in diameter)productivity versus biomass were close to 1.0 for all ecosystem types and functional groups.Scaling exponents decreased in coarse roots(>2mm in diameter).Conclusions:We found isometric metabolic scaling in fine roots,a metabolically active organ similar to seedlings or saplings.Our findings also indicate a shift in metabolic scaling during plant development.Overall,our study supports the absence of any unified single constant scaling exponent for metabolism-biomass relationships in terrestrial plants,especially for forests with woody species.

Dual-Use Bioenergy-Livestock Feed Potential of Giant Miscanthus, Giant Reed, and Miscane

High yielding perennial grasses could integrate bioenergy-livestock operations, thereby, offsetting diversions of cropland to lignocellulosic crops, but research is needed to determine chemical composition and digestibility of leaf and stem fractions that might affect downstream reside uses. The objective of this study was to compare feedstock quality of leaf and stem tissues of dedicated bioenergy feedstocks: giant miscanthus (Miscanthus × giganteus), giant reed (Arundo donax), and miscane (Saccharum hybrid × Miscanthus spp.) when grown with or without supplemental irrigation on an upland site. Three species were space-planted on a silt loam soil in March 2007 and harvested prior to the first freeze in plant-cane, first ratoon, and second-ratoon crops for three years. Giant miscanthus leaf tissue had greatest acid detergent lignin and cellulose, and lowest concentrations of nitrogen (N) and total nonstructural carbohydrates (TNC) in ratoon crops. Giant reed leaf tissue had greatest concentrations of in vitro digestible dry matter (IVDMD), TNC, and N (P ≤ 0.05). Conversely, miscane stem tissue had greatest concentrations of IVDMD, TNC, hemicellulose, and low dry matter and combustible energy (P ≤ 0.05). Results suggest all species’ residue has positive feedstock attributes for thermochemical bioenergy conversion, and albeit giant miscanthus has very little potential value as fodder. Miscane stem and giant reed leaf tissue have potential value as livestock feed, although giant reed is not currently recommended for planting. Further research is needed on dietary composition, acceptability, voluntary intake, and live weight gain before any of these species are recommended as livestock feed sources.

Transcriptional response of Microcystis aeruginosa to the recruitment promoting-benthic bacteria

Blooms of Microcystis aeruginosa occur frequently in many freshwater ecosystems around the world,but the mechanism of recovery has not been fully understood.In our previous study,three benthic bacterial species(E.sp013,Ba.spD06,and Ba.spD24)were identifi ed capable of promoting the recruitment of M.aeruginosa.Here,we further investigated the transcriptional response of M.aeruginosa to the benthic bacteria in early phase of recruitment by means of RNA-Seq analysis.In total,5803803 unigenes on average length of 404 bp were obtained from the transcriptome of M.aeruginosa.There were 54982 unigenes identifi ed as benthic bacteria-responsive unigenes based on the expression level analysis.Results of the protein-protein interaction analysis(PPI)show that the hub genes of the benthic bacteria responsive unigenes mediated network were ribosomal proteins of 30S and 50S,and the most signifi cant functional module of the network was related to the ribosome.Both the unigenes encoding the translation initiation factors(IF-2,IF-3)and elongation factors(lepA,fusA,and tufA)were up-regulated to respond benthic bacteria.Therefore,it indicates that the benthic bacteria have a positive infl uence on activating the ribosome during the early recovery stage of M.aeruginosa.

Post-Emergence Control of Windmillgrass (<i>Chloris verticillata</i>Nutt.) Using Selected Herbicides

Windmillgrass (Chloris verticillata Nutt.) is spreading from native areas, becoming problematic in established turf in Missouri, and surrounding states. Limited information is available regarding post-emergence (POST) control options for turf. The objective of this research was to assess the visual and biomass response of windmillgrass to selected herbicides approved for use in turf. In one study, a greenhouse experiment was conducted with POST application of seven selected herbicides, including dimethylamine salt of quinclorac, fenoxaprop-P-ethyl, foramsulfuron, mesotrione, sulfentrazone, and topramezone in a tank-mixture with triclopyr, as well as a pre-packed product containing thiencarbazone-methyl, foramsulfuron, and halosulfuron-methyl. Under the same experimental conditions, five additional treatments, including mesotrione or topramezone applied at label-suggested rates alone or in combination with triclopyr, in addition to triclopyr alone, were applied to a second study on tillering windmillgrass. At 4 weeks after treatment (WAT), only fenoxaprop-p-ethyl consistently resulted in a complete control of windmillgrass. No measurable regrowth of plants was observed two weeks following the initial collection of windmillgrass aboveground biomass. In the second study, combinations of mesotrione and topramezone with triclopyr resulted in 100% visual injury of windmillgrass at 4 WAT;mesotrione and topramezone alone only resulted in up to 66% visual injury. Triclopyr alone resulted in 91% injury at 4 WAT. The greatest reduction of windmillgrass biomass and least amount of regrowth resulted from mesotrione and topramezone combinations with triclopyr. Control of windmillgrass is effective with fenoxaprop-P-ethyl alone, but use of a 4-Hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor such as mesotrione and topramezone should include the addition of triclopyr.

Growth Rates of Giant Miscanthus (<i>Miscanthus</i>×<i>giganteus</i>) and Giant Reed (<i>Arundo donax</i>) in a Low-Input System in Arkansas, USA

The US Department of Energy is currently building strategies for the expansion of clean and renewable energy sources, and tall, rapidly-growing grasses such as giant miscanthus (Miscanthus × giganteus) and giant reed (Arundo donax) are two of the many of species that could fill this renewable energy niche. The objective was to compare stalk growth components of giant miscanthus and giant reed, in a low-input system (no irrigation and no fertilizer use) in Arkansas, USA. Due to the potential invasiveness of giant reed, our study was conducted on an upland site to minimize escape. Plant height and dry weight per stalk were measured every week for two consecutive growing seasons in 2012 and 2013. Leaf area index (LAI) was measured every two weeks from May to September in 2012. A significant species × day interaction occurred for plant height and dry weight per stalk, due to the relatively greater height and weight of giant reed compared to giant miscanthus after May. Stalk elongation rate was greater for giant reed than giant miscanthus (1.85 and 1.11 cm day-1, respectively). Leaf area index differed between species, giant reed (10.4 m2 m-2) > giant miscanthus (4.4 m2 m-2). We showed that giant reed produced taller, heavier stalks, and had a greater stalk elongation rate, compared to giant miscanthus. For sustainable bioenergy production from giant reed in Arkansas, further studies should be performed to determine ideal number of harvests per year and associated production cost.

Effect of Ensiling on Nutritional Properties of Sericea Lespedeza Alone or in Mixtures with Alfalfa

In this investigation,fresh sericea lespedeza(SL;Lespedeza cuneata)and alfalfa(Medicago sativa)were cut in the field,frozen,chopped and mixed into ratios of 100:0,75:25,50:50,25:75 and 0:100,respectively,with each treatment combination packed into 12 mini-silos and sealed to be air-tight.Three mini-silos per treatment were opened after 1,7,21 and 84 d of ensiling and analyzed for pH,neutral detergent fiber(NDF),acid detergent fiber(ADF),and acid detergent lignin(ADL),unbound and bound condensed tannins(CT),nitrogen(N),nitrate N(NO3-N),and ammonia N(NH4+N)content.All of the forage combinations ensiled well,with a rapid drop in pH(below 5.0 by Day 7).Fiber concentrations(NDF,ADF,ADL)were greater in 75% and 100% SL silages than in 0%,25% and 50% SL samples by Day 84 of the study,possibly due to interference of CT in the detergent analysis system.Concentrations of N,NO3-N and NH4+N were decreased in silages as percentage SL in the mixture increased,while unbound,bound and total CT increased as percentage SL increased.In this study,there was reduced proteolysis during ensiling of combinations of SL and alfalfa,as indicated by reduced NO3-N and NH4+N production as percentage SL in the silage mixtures increased.

SMRT-AgRenSeq-d in potato (Solanum tuberosum) as a method to identify candidates for the nematode resistance Gpa5

Potato is the thirdmost important food crop in theworld.Diverse pathogens threaten sustainable crop production but can be controlled,in many cases,through the deployment of disease resistance genes belonging to the family of nucleotide-binding,leucine-rich-repeat(NLR)genes.To identify effective disease resistance genes in established varieties,we have successfully established SMRT-AgRenSeq in tetraploid potatoes and have further enhanced the methodology by including dRenSeq in an approach that we term SMR-AgRenSeq-d.The inclusion of dRenSeq enables the filtering of candidates after the association analysis by establishing a presence/absence matrix across resistant and susceptible varieties that is translated into an F1 score.Using a SMRT-RenSeq-based sequence representation of the NLRome from the cultivar Innovator,SMRT-AgRenSeq-d analyses reliably identified the late blight resistance benchmark genes Rpi-R1,Rpi-R2-like,Rpi-R3a,and Rpi-R3b in a panel of 117 varieties with variable phenotype penetrations.All benchmark genes were identified with an F1 score of 1,which indicates absolute linkage in the panel.This method also identified nine strong candidates for Gpa5 that controls the potato cyst nematode(PCN)species Globodera pallida(pathotypes Pa2/3).Assuming that NLRs are involved in controlling many types of resistances,SMRT-AgRenSeq-d can readily be applied to diverse crops and pathogen systems.

基于结构方程模型的玉米施氮量-光合产物-产量关系研究

玉米是重要的粮食作物,其最终产量与吐丝期穗的发育密切相关,而玉米穗发育受到土壤水分、氮素和其他养分营养调控。但是,不同施氮水平下,玉米幼穗中碳水化合物含量如何变化,怎样影响产量及其构成因子,至今还缺乏综合分析。该研究通过2 a的田间试验,比较了3个施氮处理下(0,150和300 kg/hm2,以N计),玉米吐丝期幼穗中葡萄糖、果糖、蔗糖和淀粉含量的差异,并通过结构方程模型综合分析它们与2个重要的产量构成因子穗粒数和穗粒质量,以及最终产量的关系。结果发现,施氮显著增加了籽粒产量、穗粒数和百粒质量。在吐丝期,氮肥施用提高了幼穗干质量和穗轴上的小花原基数。同时,玉米幼穗中的葡萄糖和果糖含量随施氮量增加而增加,而蔗糖和淀粉含量随施氮量增加呈下降趋势。结构方程模型的结果显示,在不同氮水平处理下,穗粒数和百粒质量能够解释产量变异的91%,其中,穗粒数对最终产量的影响较大,标准化路径系数为0.66,而百粒质量对产量的路径系数只有0.34。同时,碳水化合物含量的变化显著影响穗粒数和百粒质量,分别解释二者变异的82%和59%。其中,单糖(葡萄糖和果糖之和)对二者的影响大致相同,标准化路径系数分别为0.47和0.52;而淀粉主要影响了穗粒数,路径系数为-0.51;相对而言,对百粒质量的影响较小,路径系数为-0.31。蔗糖含量对穗粒数和百粒质量都没有显著影响。综上,玉米吐丝期幼穗中碳水化合物含量受到土壤氮素有效性的影响,并对玉米最终产量及其构成因子具有重要的指示作用。该研究对揭示玉米产量形成对施氮的响应有一定参考价值。

Intracellular Transport of Plant Viruses： Finding the Door out of the Cell

Plant viruses are a class of plant pathogens that specialize in movement from cell to cell. As part of their arsenal for infection of plants, every virus encodes a movement protein （MP）, a protein dedicated to enlarging the pore size of plasmodesmata （PD） and actively transporting the viral nucleic acid into the adjacent cell. As our knowledge of intercellular transport has increased, it has become apparent that viruses must also use an active mechanism to target the virus from their site of replication within the cell to the PD. Just as viruses are too large to fit through an unmodified plasmodesma, they are also too large to be freely diffused through the cytoplasm of the cell. Evidence has accumulated now for the involvement of other categories of viral proteins in intracellular movement in addition to the MP, including viral proteins originally associated with replication or gene expression. In this review, we will discuss the strategies that viruses use for intracellular movement from the replication site to the PD, in particular focusing on the role of host membranes for intracellular transport and the coordinated interactions between virus proteins within cells that are necessary for successful virus spread.

Whole-Plant Live Imaging of Reactive Oxygen Species

Reactive oxygen species(ROS)are key regulators of numerous subcellular,cellular,and systemic signals.They function in plants as an integral part of many different hormonal,physiological,and developmental pathways,as well as play a critical role in defense and acclimation responses to different biotic and abiotic conditions.Although many ROS imaging techniques have been developed and utilized in plants,a wholeplant imaging platform for the dynamic detection of ROS in mature plants is lacking.Here we report a robust and straightforward method for the whole-plant live imaging of ROS in mature plants grown in soil.This new method could be used to study local and systemic ROS signals in different genetic variants,conduct phenotyping studies to identify new pathways for ROS signaling,monitor the stress level of different plants and mutants,and unravel novel routes of ROS integration into stress,growth regulation,and development in plants.We demonstrate the utility of this new method for studying systemic ROS signals in different A rabidopsis mutants and wound responses in cereals such as wheat and corn.

Kinematic Analysis of Leaf Growth in Grasses:A Comment on Spatial and Temporal Quantitative Analysis of Cell Division and Elongation Rate in Growing Wheat Leaves under Saline Conditions

Hu and Schmidhalter （2008） conducted a study with wheat seedlings growing in saline and non-stressed （control） conditions with the aim of identifying and quantifying the cellular basis for the reduction in leaf growth. We applaud their goals as salinity is an important issue for plant ecology and food production; however, we have concerns about the methodology used and the subsequent conclusJons that are drawn.

Dynamics and origin of cytokinins involved in plant manipulation by a leaf-mining insect

Several herbivorous insects and plant-associated microorganisms control the phytohormonal balance, thus enabling them to successfully exploit the plant by inhibiting plant defenses and withdrawing plant resources for their own benefit. The leaf-mining moth Phyllonorycter blancardella modifies the cytokinin （CK） profile of mined leaf-tissues, and the insect symbiotic bacteria Wolbachia is involved in the plant manipulation to the benefit of the insect host. To gain a deeper understanding into the possible origin and dynamics of CKs, we conducted an extensive characterization of CKs in larvae and in infected apple leaves. Our results show the enhanced CK levels in mines, both on green and yellow leaves, allowing insects to control their nutritional supply under fluctuating environmental conditions. The spatial distribution of CKs within the mined leaves shows that hormone manipulation is strictly limited to the mine suggesting the absence of CK translocation from distant leaf areas toward the insect feeding site. Mass spectrometry analyses reveal that major CK types accumulating in mines and larvae are similar to what is observed for most gall-inducers, suggesting that strategies underlying the plant manipulation may be shared between herbivorous insects with distinct life histories. Results further show that CKs are detected in the highest levels in larvae, reinforcing our hypothesis that CKs accumulating in the mines originate from the insect itself. Presence of bacteria- specific methylthio-CKs is consistent with previous results suggesting that insect bacterial symbionts contribute to the observed phenotype. Our study provides key findings toward the understanding of molecular mechanisms underlying this intricate plant-insect-microbe interaction.