Molecular Plant-microbe Interactions最新文献

{"title":"Generation of Inheritable A-to-G Transitions Using Adenine Base Editing and NG-PAM Cas9 in <i>Arabidopsis thaliana</i>.","authors":"Yi Yun Tan, Yin Yin Liew, Rachelle R Q Lee, Baptiste Castel, Nga Man Chan, Wei-Lin Wan, Yizhong Zhang, Donghui Hu, Persis Chan, Sang-Tae Kim, Eunyoung Chae","doi":"10.1094/MPMI-10-24-0127-TA","DOIUrl":"10.1094/MPMI-10-24-0127-TA","url":null,"abstract":"<p><p>Towards precise genome editing, base editors have been developed by fusing catalytically compromised Cas9 with deaminase components, mediating C-to-T (cytosine base editors) or A-to-G (adenine base editors) transition. We developed a set of vectors consisting of a 5'-NG-3' PAM-recognizing variant of SpCas9 with adenosine deaminases TadA7.10 or TadA8e. Using a phenotype-based screen in <i>Arabidopsis thaliana</i> targeting multiple <i>PDS3</i> intron splice sites, we achieved up to 81% somatic A-to-G editing in primary transformants at a splice acceptor site with NGG PAM, while 35% was achieved for the same target adenine with NGA PAM. Among tested vectors, pECNUS4 (Addgene #184887), carrying TadA8e, showed the highest adenine base editor (ABE) efficiency. With pECNUS4, we recreated a naturally occurring allele of <i>DANGEROUS MIX3</i> (<i>DM3</i>) in two generations, transgene-free, for NGC PAM. We also simultaneously base-edited four redundant <i>DM1/SSI4</i> homologs, encoding nucleotide-binding leucine-rich repeat (NLR) proteins, using a single gRNA with NGA PAM targeting the conserved yet functionally crucial P-loop motif of NLR proteins. We found fixation of A-to-G in three NLR genes for all three possible adenine sites within base-editing window 3-9, as the edited genes segregate in T₂. Multigene targeting succeeded in rescuing the previously reported autoimmune phenotype in two generations. Mediating desired ABE on seven NLR genes simultaneously was successful as well; above 77% editing was achieved in six of the seven possible targets in a T₁ plant, with the remaining having a moderately high (32%) editing. ABE application to specifically inactivate functional motifs is anticipated to expedite the discovery of novel roles for proteins. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.</p>","PeriodicalId":19009,"journal":{"name":"Molecular Plant-microbe Interactions","volume":" ","pages":"30-42"},"PeriodicalIF":3.2,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142716728","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Transcriptomic Profiling of '<i>Candidatus</i> Liberibacter asiaticus' in Different Citrus Tissues Reveals Novel Insights into Huanglongbing Pathogenesis.","authors":"Amelia H Lovelace, Chunxia Wang, Amit Levy, Wenbo Ma","doi":"10.1094/MPMI-08-24-0102-R","DOIUrl":"10.1094/MPMI-08-24-0102-R","url":null,"abstract":"<p><p>'<i>Candidatus</i> Liberibacter asiaticus' (Las) is a gram-negative bacterial pathogen associated with citrus huanglongbing (HLB) or greening disease. Las is transmitted by the Asian citrus psyllid (ACP) where it colonizes the phloem tissue, resulting in substantial economic losses to the citrus industry worldwide. Despite extensive efforts, effective management strategies against HLB remain elusive, necessitating a deeper understanding of the pathogen's biology. Las undergoes cell-to-cell movement through phloem flow and colonizes different tissues in which Las may have varying interactions with the host. Here, we investigate the transcriptomic landscape of Las in citrus seed coat vasculatures, enabling a complete gene expression profiling of Las genome and revealing unique transcriptomic patterns compared with previous studies using midrib tissues. Comparative transcriptomics between seed coat, midrib, and ACP identified specific responses and metabolic states of Las in different host tissue. Two Las virulence factors that exhibit higher expression in seed coat can suppress callose deposition. Therefore, they may contribute to unclogging sieve plate pores during Las colonization in seed coat vasculature. Furthermore, analysis of regulatory elements uncovers a potential role of LuxR-type transcription factors in regulating Liberibacter effector gene expression during plant colonization. Together, this work provides novel insights into the pathogenesis of the devastating citrus HLB. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.</p>","PeriodicalId":19009,"journal":{"name":"Molecular Plant-microbe Interactions","volume":" ","pages":"56-71"},"PeriodicalIF":3.2,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142583864","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Photosynthesis Responses to the Infection with Plant Pathogens.","authors":"Alissar Cheaib, Nabil Killiny","doi":"10.1094/MPMI-05-24-0052-CR","DOIUrl":"10.1094/MPMI-05-24-0052-CR","url":null,"abstract":"<p><p>Photosynthesis, the remarkable process by which green plants synthesize nutrients using light energy, plays a crucial role in sustaining life on Earth. However, the effects of pathogens on photosynthesis are not widely understood. In general, a reduction of photosynthesis occurs upon the infection with pathogens. Two main scenarios are responsible for the reduction in photosynthetic capacity. In the first scenario, the pathogen attacks green aerial tissues, such as when caused by fungal and bacterial leaf spots and blights, which affect photosynthesis by destroying green leaf tissue or causing defoliation. This leads to a decrease in the photosynthetic area, ultimately reducing photosynthesis. Interestingly, even when the overall chlorophyll content of leaves is significantly reduced due to pathogen invasion, the remaining chlorophyll-containing leaf area may maintain or even enhance its photosynthetic efficiency. This compensatory mechanism helps mitigate the loss of photosynthetic area. However, the overall yield of the plant is still affected. The second scenario is a reduction in chlorophyll content due to chlorosis, which is characterized by yellowing of leaves. It is a common symptom of plant diseases. It refers to a reduction in the amount of chlorophyll per chloroplast rather than a decrease in chloroplast number. Diseases caused by viruses and phytoplasmas often exhibit chlorosis. While pathogens disrupt photosynthesis, plants exhibit significant adaptations to cope with these challenges. Understanding these interactions is essential for sustainable agriculture and ecosystem health. Thus, in this review, we discuss the effect of several pathogens on the photosynthesis processes and efficiency in detail. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.</p>","PeriodicalId":19009,"journal":{"name":"Molecular Plant-microbe Interactions","volume":" ","pages":"9-29"},"PeriodicalIF":3.2,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142624220","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Liberibacter Turning Citrus into Its Hideout.","authors":"Ruby Tiwari","doi":"10.1094/MPMI-01-25-0007-CM","DOIUrl":"https://doi.org/10.1094/MPMI-01-25-0007-CM","url":null,"abstract":"","PeriodicalId":19009,"journal":{"name":"Molecular Plant-microbe Interactions","volume":"38 1","pages":"4-5"},"PeriodicalIF":3.2,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143516142","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Redox Status-Selective Imaging of Iron in Vegetative and Pathogenic Fungal Cells Using Fluorescent Dyes Synthesized Via Simple Chemical Reactions.","authors":"Lala Aliyeva-Schnorr, Niels V Heise, René Csuk, Holger B Deising","doi":"10.1094/MPMI-09-24-0111-SC","DOIUrl":"10.1094/MPMI-09-24-0111-SC","url":null,"abstract":"<p><p>Iron plays a prominent role in various biological processes and is an essential element in almost all organisms, including plant-pathogenic fungi. As a transition element, iron occurs in two redox states, Fe²⁺ and Fe³⁺, the transition between which generates distinct reactive oxygen species (ROS) such as H₂O₂, OH^- anions, and toxic OH· radicals. Thus, the redox status of Fe determines ROS formation in pathogen attack and plant defense and governs the outcome of pathogenic interactions. Therefore, spatially resolved visualization of Fe²⁺ and Fe³⁺ are essential to understand microbial pathogenesis. Here, we report a simple method for synthesis of the redox-state-selective dyes pyrene-tetramethyl piperidinyl oxyl (p-TEMPO) and 4-(4-methylpiperazine-1)-7-nitrobenz-2-oxa-1,3-diazole (MPNBD) for fluorescence microscopy-based imaging of Fe²⁺ and Fe³⁺ ions. Using these dyes, the occurrence and spatial distribution of Fe²⁺ and Fe³⁺ ions in vegetative and pathogenic hyphae of the hemibiotrophic maize anthracnose fungus <i>Colletotrichum graminicola</i> are shown. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.</p>","PeriodicalId":19009,"journal":{"name":"Molecular Plant-microbe Interactions","volume":" ","pages":"50-55"},"PeriodicalIF":3.2,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143046939","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PP2C Phosphatase Pic6 Suppresses MAPK Activation and Disease Resistance in Tomato.","authors":"Joydeep Chakraborty, Guy Sobol, Fan Xia, Ning Zhang, Gregory B Martin, Guido Sessa","doi":"10.1094/MPMI-10-24-0124-SC","DOIUrl":"10.1094/MPMI-10-24-0124-SC","url":null,"abstract":"<p><p>Type 2C protein phosphatases (PP2Cs) are essential for regulating plant immune responses to pathogens. Our study focuses on the tomato PP2C-immunity associated candidate 6 (Pic6), elucidating its role in negatively regulating pattern-triggered immunity (PTI) signaling pathways in tomato. Using reverse-transcription quantitative polymerase chain reaction (RT-qPCR), we observed that treatment with microbe-associated molecular patterns (MAMPs)-flg22 and flgII-28-significantly increased <i>Pic6</i> mRNA levels in wild-type (RG-PtoR) tomato plants. Pic6 features a conserved N-terminal kinase-interacting motif (KIM) and a C-terminal PP2C domain. We produced variants of Pic6 with mutations in these regions, demonstrating their involvements in negatively regulating tomato immunity. <i>Agrobacterium</i>-mediated transient overexpression of Pic6 resulted in enhanced growth of the bacterial pathogen <i>Pseudomonas syringae</i> pathovar <i>tomato</i> (<i>Pst</i>) strain DC3000Δ<i>hopQ1-1</i> compared with a yellow fluorescent protein (YFP) control. Additionally, Pic6 overexpression inhibited mitogen-activated protein kinase (MAPK) activation in response to flg22 and flgII-28 treatments. Importantly, Pic6 exhibited phosphatase activity and interacted with tomato Mkk1/Mkk2 proteins and dephosphorylated them in a KIM-dependent manner. Furthermore, we generated RG-pic6 loss-of-function mutants by CRISPR/Cas9, revealing that the absence of Pic6 heightened MAPK activity and increased resistance to <i>Xanthomonas euvesicatoria</i> strain 85-10 (<i>Xe</i> 85-10) when compared with the wild-type (RG-PtoR) plants. Transcript analyses showed that after flg22/flgII-28 treatment, PTI-reporter genes <i>NAC</i> and <i>Osmotin</i> were significantly upregulated in RG-pic6 mutants in comparison to the wild-type (RG-PtoR) plants. Overall, our findings indicate that Pic6 acts as a negative regulator of MAPK signaling and plays a pivotal role in modulating tomato immunity against bacterial pathogens. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.</p>","PeriodicalId":19009,"journal":{"name":"Molecular Plant-microbe Interactions","volume":" ","pages":"43-49"},"PeriodicalIF":3.2,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142644582","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Differential Transcriptome Reprogramming Induced by the Soybean Cyst Nematode Type 0 and Type 1.2.5.7 During Resistant and Susceptible Interactions.","authors":"Mst Shamira Sultana, Daniel Niyikiza, Tracy E Hawk, Nicole Coffey, Valéria Lopes-Caitar, Alexander C Pfotenhauer, Hana El-Messidi, Chris Wyman, Vince Pantalone, Tarek Hewezi","doi":"10.1094/MPMI-08-24-0092-R","DOIUrl":"10.1094/MPMI-08-24-0092-R","url":null,"abstract":"<p><p>Soybean cyst nematode (SCN, <i>Heterodera glycines</i> [Hg]) is a serious root parasite of soybean (<i>Glycine max</i>) that induces extensive gene expression changes associated with pleiotropic biological activities in infected cells. However, the impacts of various SCN Hg types on host transcriptome reprogramming remain largely unknown. Here, we developed and used two recombinant inbred lines (RIL; RIL-72 and RIL-137) to profile transcriptome reprogramming in the infection sites during the resistant and susceptible interactions with SCN Hg Type 1.2.5.7 and Type 0. SCN bioassays indicated that RIL-72 was susceptible to Type 1.2.5.7 but resistant to Type 0, whereas RIL-137 was resistant to both types. Comparative analysis of gene expression changes induced by Type 1.2.5.7 in the resistant and susceptible lines revealed distinct transcriptome regulation with a number of similarly and oppositely regulated genes. The expression levels of similarly regulated genes in the susceptible line appeared to be insufficient to mount an effective defense against SCN. The functional importance of oppositely regulated genes was confirmed using virus-induced gene silencing (VIGS) and overexpression approaches. Further transcriptome comparisons revealed shared as well as Hg type- and genotype-specific transcriptome reprogramming. Shared transcriptome responses were mediated through common SCN-responsive genes and conserved immune signaling, whereas genotype-specific responses were derived from genetic variability, metabolic and hormonal differences, and varied regulation of protein phosphorylation and ubiquitination. The conserved defense mechanisms together with genotype-specific responses would enable plants to trigger effective and tailored immune responses to various Hg types and adapt the defense response to their genetic backgrounds. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.</p>","PeriodicalId":19009,"journal":{"name":"Molecular Plant-microbe Interactions","volume":" ","pages":"828-840"},"PeriodicalIF":3.2,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400810","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"miR472 Deficiency Enhances <i>Arabidopsis thaliana</i> Defense Without Reducing Seed Production.","authors":"Francois Vasseur, Patricia Baldrich, Tamara Jiménez-Góngora, Luis Villar-Martin, Detlef Weigel, Ignacio Rubio-Somoza","doi":"10.1094/MPMI-02-24-0011-R","DOIUrl":"10.1094/MPMI-02-24-0011-R","url":null,"abstract":"<p><p>After having co-existed in plant genomes for at least 200 million years, the products of microRNA (miRNA) and nucleotide-binding leucine-rich repeat protein (NLR) genes formed a regulatory relationship in the common ancestor of modern gymnosperms and angiosperms. From then on, DNA polymorphisms occurring at miRNA target sequences within NLR transcripts must have been compensated by mutations in the corresponding mature miRNA sequence. The potential evolutionary advantage of such regulation remains largely unknown and might be related to two nonexclusive scenarios: (i) miRNA-dependent regulation of NLR levels might prevent defense mis-activation with negative effects on plant growth and reproduction or (ii) reduction of active miRNA levels in response to pathogen-derived molecules (pathogen-associated molecular patterns [PAMPs] and silencing suppressors) might rapidly release otherwise silent NLR transcripts for rapid translation and thereby enhance defense. Here, we used <i>Arabidopsis thaliana</i> plants deficient for miR472 function to study the impact of releasing its NLR targets on plant growth and reproduction and on defense against the fungal pathogen <i>Plectosphaerella cucumerina</i>. We show that miR472 regulation has a dual role, participating both in the tight regulation of plant defense and growth. MIM472 lines, with reduced active miR472, are more resistant to pathogens and, correlatively, have reduced relative growth compared with wild-type plants, although the end of their reproductive phase is delayed, exhibiting higher adult biomass and similar seed yield as the wild-type. Our study highlights how negative consequences of defense activation might be compensated by changes in phenology and that miR472 reduction is an integral part of plant defense responses. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.</p>","PeriodicalId":19009,"journal":{"name":"Molecular Plant-microbe Interactions","volume":" ","pages":"819-827"},"PeriodicalIF":3.2,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142350547","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evolutionary and Epidemiological Insights from Historical and Modern Genomes of <i>Xanthomonas oryzae</i> pv. <i>oryzicola</i>, the Causal Agent of Bacterial Leaf Streak of Rice.","authors":"Mathilde Hutin, Sara Carpenter, Shivrajani Baruah, Paola Campos, Karine Boyer, Dodelys Andriantsimialona, Solo Hery Rapanarivo, Olivier Pruvost, Nathalie Becker, Lionel Gagnevin, Ralf Koebnik, Boris Szurek, Ousmane Koita, Adam J Bogdanove, Adrien Rieux","doi":"10.1094/MPMI-05-24-0062-SC","DOIUrl":"10.1094/MPMI-05-24-0062-SC","url":null,"abstract":"<p><p><i>Xanthomonas oryzae</i> pv. <i>oryzicola</i> (<i>Xoc</i>) causes bacterial leaf streak (BLS) of rice. This disease represents a major constraint for rice production, which is a crop feeding more than half of the world's population. <i>Xoc</i> was first described in 1918 in the Philippines and is prevalent in southeast Asia. Today, BLS is also omnipresent in both East- and West-Africa, where the disease was first reported in the early 1980s. The appearance of <i>Xoc</i> in Africa decades after its first report in Asia suggests that the disease could have been introduced from Asia to Africa. Strict conservation of five transcription activator-like (TAL) effectors in whole-genome sequences of 10 strains of <i>Xoc</i> including three from West-Africa and seven from Asia also support this hypothesis. East-Africa, especially Madagascar, where the disease was first described in 1985 is located at the interface between Asia and Africa, hence representing an interesting region to explore the link between strains from Asia and West-Africa. In this study, we did the following: (i) reconstructed the genome of a historical <i>Xoc</i> strain from a herbarium specimen of rice showing symptoms of BLS that was sampled in Madagascar in 1931, 50 years before the first description of the disease, and (ii) sequenced nine new modern strains, including five from Madagascar and East-Africa. The analysis of those new genomes along with previously published ones shed light within the evolutionary and epidemiological history of <i>Xoc</i>. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.</p>","PeriodicalId":19009,"journal":{"name":"Molecular Plant-microbe Interactions","volume":" ","pages":"814-818"},"PeriodicalIF":3.2,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291744","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rethinking Growth and Defense: miR472 as a Sensor and Regulator of Plant Immunity.","authors":"Jawahar Singh, Ved Prakash","doi":"10.1094/MPMI-12-24-0157-CM","DOIUrl":"https://doi.org/10.1094/MPMI-12-24-0157-CM","url":null,"abstract":"","PeriodicalId":19009,"journal":{"name":"Molecular Plant-microbe Interactions","volume":"37 12","pages":"802-803"},"PeriodicalIF":3.2,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142854887","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}