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Engineered biocontainable RNA virus vectors for non-transgenic genome editing across crop species and genotypes

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Molecular Plant 2023年第3期16卷 616-631页

作者： Qian Liu Chenglu Zhao Kai Sun Yinlu Deng Zhenghe Li State Key Laboratory of Rice biology Institute of BiotechnologyZhejang UniversityHangzhouChina Ministry of Agricutture Key Laboratory of Molecular biology of Crop pathogens and Insect Pests Zhejiang UniversityHangzhouChina Key Laboratory of biology of Crop pathogens and Insects of Zhejang Province Zhejang UniversityHangzhouChina

CRISPR/Cas genome-editing tools provide unprecedented opportunities for basic plant biology research and crop breeding.However,the lack of robust delivery methods has limited the widespread adoption of these revolutionary technologies in plant science.Here,we report an efficient,non-transgenic CRISPR/Cas delivery platform based on the engineered tomato spotted wilt virus(TSWV),an RNA virus with a host range of over 1000 plant species.We eliminated viral elements essential for insect transmission to liberate genome space for accommodating large genetic cargoes without sacrificing the ability to infect plant hosts.The resulting non-insect-transmissible viral vectors enabled effective and stable in planta delivery of Cas12a and Cas9 nucleases as well as adenine and cytosine base editors.In systemically infected plant tissues,the deconstructed TSWV-derived vectors induced efficient somatic gene mutations and base conversions in multiple crop species with little genotype dependency.Plants with heritable,bi-allelic mutations could be readily regenerated by culturing the virus-infected tissues in vitro without antibiotic selection.Moreover,we showed that antiviral treatment with ribavirin during tissue culture cleared the viral vectors in 100%of regenerated plants and further augmented the recovery of heritable mutations.Because many plants are recalcitrant to stable transformation,the viral delivery system developed in this work provides a promising tool to overcome gene delivery bottlenecks for genome editing in various crop species and elite varieties.

关键词： genome editing CRISPR-Cas base editor RNA virus vector tomato spotted wilt virus bunyavirus delivery non-transgenic

Role of conspecifics and personal experience on behavioral avoidance of contaminated flowers by bumblebees

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Current Zoology 2019年第4期65卷 447-455页

作者： Bertrand Fouks Emily G. Robb H. Michael G. Lattorff lnstitut fur Biologie Molekulare Okologie Martin-Luther-Universitat Halle-Wittenberg Hoher Weg Halle (Saale) Germany Centre for Ecological and Evolutionary Synthesis (CEES) Department of Biosciences University of Oslo Oslo Norway Microbes and Pathogens Biology The Institute for Global Food Security School of Biological Sciences Queen's University Belfast United Kingdom lnternational Centre of Insect Physiology and Ecology (ICIPE) Nairobi Kenya German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig Deutscher Platz Leipzig Germany

Pollinators use multiple cues whilst foraging in eluding direct cues from flowers and in direct cues from other pollinators. The use of indirect social cues is common in social in sects, such as honeybees and bumblebees, where a social environment facilitates the ability to use such cues. Bumblebees use cues to forage on flowers according to previous foraging experiences. Flowers are an essential food source for pollinators but also pose a high risk of parasite infection through the shared use of flowers leading to parasite spillover. Nevertheless, bumblebees have evolved behavioral defense mechanisms to limit parasite infection by avoiding contaminated flowers. Mechanisms underlying the avoidance of contaminated flowers by bumblebees are poorly understood. Bumblebees were recorded having the choice to forage on non-contaminated flowers and flowers contaminated by a trypan osome gut parasite, Crithidia bombi. The use of different treatments with presence or absence of con specifics on both con taminated and non-contami nated flowers allowed to investigate the role of social visual cues on their pathogen avoidance behavior. Bumblebees are expected to use social visual cues to avoid contaminated flowers. Our study reveals that the presence of a con specific on flowers either con taminated or not does not help bumblebee foragers avoiding contaminated flowers. Nevertheless, bumblebees whereas gaining experie nee tend to avoid their con specific when placed on contami nated flower and copy it whe n on the non-contami nated flower. Our experime nt suggests a detrime ntal impact of floral see nt on disease avoidanee behavior.

关键词： Host-parasite in teractions social learning pollinators inadvertent social information behavioral immunity copying behavior

Cu(Ⅱ), Fe(Ⅲ) and Mn(Ⅱ) combinations as environmental stress factors have distinguishing effects on Enterococcus hirae

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Journal of Environmental Sciences 2015年第2期27卷 95-100页

作者： Zaruhi Vardanyan Armen Trchounian Department of Biophysics Faculty of Biology Yerevan State University Department of Microbiology Plants and Microbes Biotechnology Faculty of Biology Yerevan State University

Pollution by various heavy metals as environmental stress factors might affect bacteria. It was established that iron（Fe（Ⅲ））, manganese（Mn（Ⅱ）） and copper（Cu（Ⅱ）） ion combinations caused effects on Enterococcus hirae that differed from the sum of the effects when the metals were added separately. It was shown that the Cu2＋–Fe3＋combination decreased the growth and ATPase activity of membrane vesicles of wild-type E. hirae ATCC9790 and atp D mutant（with defective FoF1-ATPase） MS116. Addition of Mn2＋–Fe3＋combinations within the same concentration range had no effects on growth compared to control（without heavy metals）. ATPase activity was increased in the presence of Mn2＋–Fe3＋, while together with0.2 mmol/L N,N′-dicyclohexylcarbodiimide（DCCD）, ATPase activity was decreased compared to control（when only 0.2 mmol/L DCCD was present）. These results indicate that heavy metals ion combinations probably affect the FOF1-ATPase, leading to conformational changes. Moreover the action may be direct or be mediated by environment redox potential.The effects observed when Fe3＋was added separately disappeared in both cases, which might be a result of competing processes between Fe3＋and other heavy metals. These findings are novel and improve the understanding of heavy metals ions effects on bacteria,and could be applied for regulation of stress response patterns in the environment.

关键词： Heavy metal ions Environmental stress Bacterial growth ATPase activity Enterococci

The receptor-like cytosolic kinase RIPK activates NADP-malic enzyme 2 to generate NADPH for fueling ROS production

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Molecular Plant 2022年第5期15卷 887-903页

作者： Binyan Wu Ping Li Xiufang Hong Cuihong Xu Ran Wang Yan Liang Ministry of Agriculture Key Laboratory of Molecular biology of Crop pathogens and Insects Institute of BiotechnologyZhejiang UniversityHangzhou 310058China

Reactive oxygen species(ROS)production is a conserved immune response in Arabidopsis primarily medi-ated by respiratory burst oxidase homolog D(RBOHD),a nicotinamide adenine dinucleotide phosphate(NADPH)oxidase associated with the plasma membrane.A rapid increase in NADPH is necessary to fuel RBOHD proteins and thus maintain ROS production.However,the molecular mechanism by which NADPH is generated to fuel RBOHD remains unclear.In this study,we isolated a new mutant allele of FLAGELLIN-INSENSITIVE 4(FIN4),which encodes the first enzyme in de novo NAD biosynthesis.fin4 mu-tants show reduced NADPH levels and impaired ROS production.However,FIN4 and other genes involved in NAD-and NADPH-generating pathways are not highly upregulated upon elicitor treatment,raising a pos-sibility that a cytosolic NADP-linked dehydrogenase might be post-transcriptionally activated to maintain the NADPH supply close to RBOHD.To verify this possibility,we isolated the proteins associated with RPM1-INDUCED PROTEIN KINASE(RIPK),a receptor-like cytoplasmic kinase that regulates broad-spectrum ROS signaling in plant immunity,and identified NADP-malic enzyme 2(NADP-ME2),an NADPH-generating enzyme.Compared with wild-type plants,nadp-me2 mutants display decreased NADP-ME activity,lower NADPH levels,and reduced ROS production in response to immune elicitors.Furthermore,we found that RIPK can directly phosphorylate NADP-ME2 and enhance its activity in vitro.The phosphorylation of the NADP-ME2 S371 residue contributes to ROS production upon immune elicitor treatment and susceptibility to the necrotrophic bacterium Pectobacterium carotovorum.Collectively,our study suggests that RIPK phosphorylates and activates NADP-ME2 to rapidly increase cytosolic NADPH,thus fueling RBOHD to sustain ROS production in plant immunity.

关键词： NADP-ME2 NADPH RBOHD reactive oxygen species plant immunity

Moth sex pheromones affect interspecific competition among sympatric species and possibly population distribution by modulating pre-mating behavior

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Insect Science 2023年第2期30卷 501-516页

作者： Jianjun Cheng Qinghua Chen Qianshuang Guo Yongjun Du Ministry of Agriculture Key Lab of Molecular biology of Crop pathogens and Insects Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang ProvinceZhejiang UniversityHangzhouChina Institute of Plant Protection Sichuan Academy of Agricultural Sciences/Key Laboratory of Integrated Pest Management on Crops in SouthwestMinistry of AgricultureChengduChina

Premating behaviors mediated by pheromones play pivotal roles in animal mating choices.In natural populations of the striped stem borer Chilo suppressalis and the rice leaf roller Cnaphalocrocis medinalis in the rice field habitat,we discovered that Z11-16:Ald,a major component of the C.suppressalis pheromone,modulated the premating behavior of C.medinalis.Z11-16:Ald evoked a strong olfactory response in male antennae and strongly inhibited the sex pheromone trapping of male C.medinalis in the field.The functions of three C.medinalis sex pheromone receptor genes(CmedPR1–3)were verified through heterologous expression in Xenopus oocytes.CmedPR1 responded to Z11-18:OH and Z11-18:Ald,as well as the interspecific pheromone compound Z11-16:Ac of sympatric species;CmedPR2 responded to Z13-18:OH and Z13-18:Ald,as well as the sex pheromone compounds Z11-16:Ald and Z9-16:Ald of sympatric species;and CmedPR3 responded to Z11-18:OH and Z13-18:OH,as well as the interspecific pheromones Z11-16:OH,Z9-16:Ald,Z11-16:Ac,and Z11-16:Ald of sympatric species.Thus,CmedPR2 and CmedPR3 share the ligand Z11-16:Ald,which is not a component of the C.medinalis sex pheromone.Therefore,the sex pheromones of interspecific species affected the input of neural signals by stimulating the sex pheromone receptors on the antennae of male C.medinalis moths,thereby inhibiting the olfactory responses of the male moths to the sex pheromones.Our results demonstrate chemical communication among sympatric species in the rice field habitat,the recognition of intra-and interspecific sex pheromones by olfactory receptors,and how insect premating behaviors are modulated to possibly affect resource partitioning.

关键词： Chilo suppressalis Cnaphalocrocis medinalis premating sex pheromone sex pheromone receptors two-electrode voltage-clamp

R_(SC3)K of soybean cv. Kefeng No.1 confers resistance to soybean mosaic virus by interacting with the viral protein P3

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Journal of Integrative Plant biology 2023年第3期65卷 838-853页

作者： Tongtong Jin Jinlong Yin Tao Wang Song Xue Bowen Li Tingxuan Zong Yunhua Yang Hui Liu Mengzhuo Liu Kai Xu Liqun Wang Guangnan Xing Haijian Zhi Kai Li National Key L aboratory for Crop Genetics and Germplasm Enhancement Key Laboratory of Biology and Genetic Improvement of Soybean-Ministry of AgricultureNational Center for Soybean ImprovementNanjing Agricultural UniversityNanjing 210095China Jiangsu Key Laboratory for microbes and Functional Genomics College of Life SciencesNanjing Normal UniversityNanjing 210023China

Soybean mosaic virus(SMV) is one of the most devastating viral pathogens of soybean(Glycine max(L.) Merr). In total, 22 Chinese SMV strains(SC1–SC22) have been classified based on the responses of 10 soybean cultivars to these pathogens. However, although several SMVresistance loci in soybean have been identified, no gene conferring SMV resistance in the resistant soybean cultivar(cv.) Kefeng No.1 has been cloned and verified. Here, using F_(2)-derived F_(3)(F_(2:3)) and recombinant inbred line(RIL) populations from a cross between Kefeng No.1 and susceptible soybean cv. Nannong 1138-2, we localized the gene in Kefeng No.1 that mediated resistance to SMV-SC3 strain to a 90-kb interval on chromosome 2. To study the functions of candidate genes in this interval, we performed Bean pod mottle virus(BPMV)-induced gene silencing(VIGS). We identified a recombinant gene(which we named R_(SC3)K) harboring an internal deletion of a genomic DNA fragment partially flanking the LOC100526921 and LOC100812666 reference genes as the SMV-SC3 resistance gene.By shuffling genes between infectious SMV DNA clones based on the avirulent isolate SC3 and virulent isolate 1129, we determined that the viral protein P3 is the avirulence determinant mediating SMV-SC3 resistance on Kefeng No.1. P3 interacts with RNase proteins encoded by R_(SC3)K, LOC100526921, and LOC100812666. The recombinant R_(SC3)K conveys much higher anti-SMV activity than LOC100526921 and LOC100812666, although those two genes also encode proteins that inhibit SMV accumulation, as revealed by gene silencing in a susceptible cultivar and by overexpression in Nicotiana benthamiana. These findings demonstrate that R_(SC3)K mediates the resistance of Kefeng No.1 to SMV-SC3 and that SMV resistance of soybean is determined by the antiviral activity of RNase proteins.

关键词： recombination resistance gene soybean mosaic virus virulence determinant

Characterization of the chemical fungicides-responsive and bacterial pathogen-preventing Bacillus licheniformis in rice spikelet

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Food Quality and Safety 2023年第2期7卷 256-266页

作者： Chengfang Zhan Mengchen Wu Hongda Fang Xiaoyu Liu Jiuyue Pan Xiaoyan Fan Mengcen Wang Haruna Matsumoto State Key Laboratory of Rice biology&Ministry of Agricultural and Rural Affairs Laboratory of Molecular biology of Crop pathogens and Insects Zhejiang UniversityHangzhouChina Key Laboratory of biology of Crop pathogens and Insects of Zhejiang Province Institute of Pesticide and Environmental ToxicologyCollege of Agriculture and BiotechnologyZhejiang UniversityHangzhouChina Zhejiang Laboratory for Systems&Precision Medicine Zhejiang University Medical CenterHangzhouChina School of Medicine Zhejiang UniversityHangzhouChina College of Plant Protection Hunan Agricultural UniversityHunanChina Plant Genetics TUM School of Life SciencesTechnical University of MunichFreisingGermany Global Education Program for AgriScience Frontiers Graduate School of AgricultureHokkaido UniversitySapporoJapan

Seed-borne bacterial pathogens cause severe yield loss and biotoxin contamination in rice,leading to increasing concern on the global food supply and environmental safety.Plant native microbes play an important role in defending against diseases,but their actions are often influ-enced by the chemical fungicides applied in the field.Here,Bacillus licheniformis mmj was isolated from rice spikelet,which uniquely showed not only fungicide-responsiveness but also broad-spectrum antimicrobial activity against major rice bacterial pathogens including Xanthomonas oryzae pv.oryzae,Burkholderia plantari and Burkholderia glumae.To understand the hallmark underlying the environmental adaptation and anti-microbial activity of B.licheniformis mmj,the genome sequence was determined by SMRT and subjected to bioinformatics analysis.Genome sequence analysis enabled the identification of a set of antimicrobial-resistance and antibacterial activity genes together with an array of harsh environment-adaptive genes.Moreover,B.licheniformis mmj metabolites were analyzed with gas chromatography coupled to triple quadrupole mass spectrometry,and the volatile components that were linked with the antimicrobial activity were preliminarily profiled.Collectively,the present findings reveal the genomic and metabolic landscapes underlying fungicide-responsive B.licheniformis,which offers a new opportunity to design harsh environment-adaptive biopesticides to cope with prevalent bacterial phytopathogens.

关键词： Bacillus licheniformis fungicide adaptation antimicrobial activity genomics metabolites rice production

Differential efficiency of a begomovirus to cross the midgut of different species of whiteflies results in variation of virus transmission by the vectors

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Science China(Life Sciences) 2018年第10期61卷 1254-1265页

作者： Lilong Pan Qunfang Chen Tao Guo Xinru Wang Ping Li Xiaowei Wang Shusheng Liu Ministry of Agriculture Key Laboratory of Molecular biology of Crop pathogens and Insects Institute of Insect Sciences Zhejiang University

Begomoviruses are important crop viral disease agents, and they are transmitted by whiteflies of the Bemisia tabaci complex.Although the transmission of begomoviruses by whiteflies has been studied for many years, the mechanisms governing differential transmission of begomoviruses by different species of the Bemisia tabaci complex remain largely unknown. Here we firstly compared the transmission efficiency of tobacco curly shoot virus(TbCSV) by four species of the B. tabaci complex and found that Asia II 1 transmitted this virus with the highest efficiency, whereas MEAM1 transmitted it with the lowest. Next, by performing quantitative analysis of virus and immune-fluorescence detection, we found that the efficiency of TbCSV to cross the midgut wall was higher in Asia II 1 than in MEAM1. Finally, we set the quantities of virions in the haemolymph to the same level in Asia II 1 and MEAM1 via injection and then compared their capacity in TbCSV transmission, and found that the difference in TbCSV transmission between them became smaller. Taken together, our findings suggest that the efficiency of a begomovirus to cross the midgut wall of a whitefly to reach the vector's haemolymph plays a significant role in determining transmission of the virus.

关键词：病毒传播向量微分种类定量分析调查结果活动范围建筑群

CLP gene family,a new gene family of Cotesia vestalis bracovirus inhibits melanization of Plutella xylostella hemolymph

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Insect Science 2021年第6期28卷 1567-1581页

作者： Ze-Hua Wang Yue-Nan Zhou Xi-Qian Ye Xiao-Tong Wu Pei Yang Min Shi Jian-Hua Huang Xue-Xin Chen Institute of Insect Science College of Agriculture and BiotechnologyZhejiang UniversityHangzhouChina Ministry of Agriculture Key Lab of Molecular biology of Crop pathogens and Insects Zhejiang UniversityHangzhouChina Zhejiang Provincial Key Lab of biology of Crop pathogens and Insects Zhejiang UniversityHangzhouChina State Key Lab of Rice biology Zhejiang UniversityHangzhouChina

Polydnaviruses(PDVs)are obligatory symbionts of parasitoid wasps and play an important role in suppressing host immune defenses.Although PDV genes that inhibit host melanization are known in Microplitis bracovirus,the functional homologs in Cotesia bracoviruses remain unknown.Here,we find that Cotesia vestalis bracovirus(CvBV)can inhibit hemolymph melanization of its host,Plutella xylostella larvae,during the early stages of parasitization,and that overexpression of highly expressed CvBV genes reduced host phenoloxidase activity.Furthermore,CvBV-7-I in particular reduced host phenolox-idase activity within 12 h,and the injection of anti-CvBV-7-1 antibody increased the melanization of parasitized host larvae.Further analyses showed that CvBV-7-1 and three homologs from other Cotesia bracoviruses possessed a C-terminal leucine/isoleucine-rich region and had a similar flinction in inhibiting melanization.Therefore,a new family of bracovirus genes was proposed and named as C-terminal Leucine/isoleucine-rich Protein(CLP).Ectopic expression of CvBV-7-1 in Drosophila hemocytes increased susceptibility to bacterial repression of melanization and reduced the melanotic encapsulation of par-asitized D.melanogaster by the parasitoid Leptopilina boulardi.The formation rate of wasp pupae and the eclosion rate of C.vestalis were affected when the function of CvBV-7-1 was blocked.Our findings suggest that CLP genes from Cotesia bracoviruses encoded proteins that contain a C-terminal leucine/isoleucine-rich region and function as melanization inhibitors during the early stage of parasitization,which is important for successful parasitization.

关键词： host immunity immunosuppression new gene family Plutella xylostella polydnavirus wasp