Project description:Seed developmental arrest is one of the early phenotypes of seed abortion. However, the molecular mechanism underlying seed developmental arrest of citrus is still unclear. In this study, laser capture microdissection (LCM) was used to accurately divide the seeds of seedless Ponkan ‘Huagan No.4’ (Citrus reticulata) (HG) and seeded Ponkan ‘Egan No.1’ (Citrus reticulata) (EG) into nucellus and integument/seed coat tissues. The captured tissues were used for subsequent RNA-seq. Moreover, single-molecule real-time (SMRT) sequencing was used to generate full-length transcripts of EG, which were used as reference transcripts for RNA-seq. These data can be utilized to analyse the causes of citrus seedlessness formation and the molecular regulatory network in the process of seed abortion.
Project description:Seed abortion is one of the important reasons for the formation of seedless characters in citrus. However, the molecular mechanism of seed abortion in citrus is still unclear. We identified several genes that may play an important role in seed abortion of 'Huagan 4', such as CrWRKY74. The downstream regulatory network of CrWRKY74 was identified based on DAP seq analysis to gain insight into the molecular mechanism related to seed abortion.
Project description:Huanglongbing (HLB) is a worldwide devastating disease of citrus. There are no effective control measures for this newly emerging but century-old disease. A powerful oligonucleotide microarray of high-density 16S rRNA genes, the PhyloChip microarray, has been developed and effectively used to study bacterial diversity, especially from environmental samples. In this article, we aim to decipher the bacterial microbiome in HLB-affected citrus versus non-infected citrus as well as in citrus plants treated with ampicillin and gentamicin using PhyloChip-based metagenomics.
Project description:‘Candidatus 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 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 from source to sink tissues and these tissues have varying responses to Las infection. Here, we investigate the transcriptomic landscape of Las in citrus sink tissues, particularly seed coat vasculatures, revealing more complete gene expression profiling of Las and unique transcriptomic pattern compared to previous studies using midrib tissues. Comparative transcriptomics between citrus seed coat vasculature and midrib tissues and ACP allowed the identification of tissue-specific responses and metabolic states of Las in planta. Furthermore, analysis of putative regulatory elements uncovers the potential role of LuxR-type transcription factors in regulating the expression of effector genes in citrus. We characterized two Las virulence factors that exhibit increased expression in seed coat vasculature tissue. Their ability to suppress different immune outputs was accessed and we demonstrate that they may contribute to the suppression of callose deposition during Las colonization. Moreover, this work provides novel insights into the pathogenesis of the devastating citrus HLB.
Project description:Huanglongbing (HLB) is a worldwide devastating disease of citrus. There are no effective control measures for this newly emerging but century-old disease. A powerful oligonucleotide microarray of high-density 16S rRNA genes, the PhyloChip microarray, has been developed and effectively used to study bacterial diversity, especially from environmental samples. In this article, we aim to decipher the bacterial microbiome in HLB-affected citrus versus non-infected citrus as well as in citrus plants treated with ampicillin and gentamicin using PhyloChip-based metagenomics. The antibiotic treatments were conducted on the randomized complete block design with three replicates. For each replicate, 15 scions were treated in each antibiotic treatment (Amp and Gm) and control (CK1 and CK2). HLB-affected budsticks were sampled from severely HLB-affected field rough lemons (cv. Lemon #76) at the USDA-ARS-USHRL farm in Fort Pierce, FL and tested positive for Las by real-time qPCR. They were soaked in the antibiotic treatments; ampicillin sodium at a concentration of 1.0 g/L (Amp, Sigma-Aldrich, St. Louis, MO) or gentamicin sulfate at a concentration of 100 mg/L (Gm, Sigma-Aldrich, St. Louis, MO) and water as the diseased control (CK1), overnight in a fume hood under ventilation and lighting. Las-free budsticks, which tested negative by qPCR from healthy rough lemons, were also soaked in water as the healthy control (CK2). The budsticks were grafted onto two-year-old healthy grapefruit (Citrus paradisi 'Duncan') rootstocks and covered using plastic tape for three weeks. To improve scion growth, new flush from the rootstocks was removed after grafting and then allowed to grow. All experimental plants were grown in an insect-proof greenhouse. The first leaf samples from scions (rough lemon) and rootstocks (grapefruit) for DNA extraction were taken four months after inoculation, and second samplings were taken at six month after inoculation. The leaves were washed in tap water and then rinsed three times with sterile water. The midribs of the leaves were excised, frozen in liquid nitrogen, and stored at -80M-BM-0C. The midribs of five leaves from each sample were pooled, and DNA was isolated for qPCR analysis for Las bacterium. DNA from the leaf midribs of scions for the PhyloChipT G3 analysis, which was extracted from all samples of the same treatment, was pooled in equal amounts and quantified by the PicoGreenM-BM-. method. The PhyloChipTM G3 analysis was conducted by Second Genome Inc. (San Francisco, CA).
Project description:Ovule developmental arrest is one of the early phenotypes of seed abortion. However, the molecular mechanism underlying ovule developmental arrest of citrus is still unclear. In this study, laser capture microdissection (LCM) was used to accurately divide the ovules of seedless Ponkan ‘Huagan NO.4’ (Citrus reticulata) (MT) and seeded Ponkan ‘Egan NO.1’ (Citrus reticulata) (WT) into nucellus and integument tissues. The captured tissues were used for subsequent RNA-seq. Moreover, single-molecule real-time (SMRT) sequencing was used to generate full-length transcripts of WT, which were used as reference transcripts for RNA-seq. These data can be utilized to analyse the causes of citrus seedlessness formation and the molecular regulatory network in the process of ovule abortion.