Project description:Citrus greening or huanglongbing (HLB) is a devastating disease of citrus. HLB is associated with the phloem-limited fastidious prokaryotic alpha-proteobacterium Candidatus Liberibacter spp. In this report, we used sweet orange (Citrus sinensis) leaf tissue infected with 'Ca. Liberibacter asiaticus' and compared this with healthy controls. Investigation of the host response was examined with citrus microarray hybridization based on 30,171 sets expressed sequence tag sequences from several citrus species and hybrids. The microarray analysis indicated that HLB infection significantly affected expression of 624 genes whose encoded proteins were categorized according to function. The categories included genes associated with sugar metabolism, plant defense, phytohormone, and cell wall metabolism, as well as 14 other gene categories.
Project description:Citrus Huanglongbing (HLB, or greening) is one of the most severe diseases of citrus. Plant disease symptom development is considered to be the consequence of a number of molecular, cellular and physiological changes, and may also be associated with host defense responses. Understanding citrus host response to HLB may contribute to the development of new strategies to control this destructive disease. We performed microarray analysis to identify the differentially expressed genes in sweet orange in response to HLB infection using the Affymetrix GeneChip® citrus genome array.
Project description:Citrus greening or huanglongbing (HLB) is a devastating disease of citrus. HLB is associated with the phloem-limited fastidious prokaryotic alpha-proteobacterium Candidatus Liberibacter spp. In this report, we used sweet orange (Citrus sinensis) leaf tissue infected with 'Ca. Liberibacter asiaticus' and compared this with healthy controls. Investigation of the host response was examined with citrus microarray hybridization based on 30,171 sets expressed sequence tag sequences from several citrus species and hybrids. The microarray analysis indicated that HLB infection significantly affected expression of 624 genes whose encoded proteins were categorized according to function. The categories included genes associated with sugar metabolism, plant defense, phytohormone, and cell wall metabolism, as well as 14 other gene categories. Young, healthy Valencia sweet orange (C. sinensis) plants were graft inoculated with budwood from Ca. L. asiaticus-infected citrus plants. Prior to the innocualtion, the plants were confirmed to be Ca. L. asiaticus-free in ordinary and quantitative PCR tests. The presence of the bacteria in the inoculated plants was confirmed in both conventional and quantitative PCR with specific primers to Ca. L. asiaticus. The stem and root samples used for RNA extraction and hybridization on Affymetrix microarrays were obtained from three symptomatic and three healthy control trees of similar size, approximately 1 year after inoculation.
Project description:Candidatus Liberibacter asiaticus (Las) is an emergent bacterial pathogen associated with the devastating citrus Huanglongbing (HLB), also known as the greening disease. Vectored by the Asian Citrus Psyllid (Diaphorina citri), Las colonizes the phloem tissue of citrus. So far, efforts of cultivating Las in vitro have not been successful and dual-transcriptome analyses could only detect ~100 Las genes due to the low abundance of bacterial RNA in infected citrus/psyllid tissues. Therefore, the biology of this pathogen is poorly understood. Here, we established a procedure to enrich Las RNA for transcriptome analysis in order to obtain insights into the interactions of Las with its two hosts. We were able to confidently determine the expression profiles of >400 Las genes, including 106 that were differentially expressed between citrus and psyllids. Genes related to transcription/translation and defense were found to be upregulated in citrus; whereas genes upregulated in psyllids are involved in metabolic pathways related to tricarboxylic acid (TCA) cycle. Genes encoding the succinate dehydrogenase and NADH quinone oxidoreductase complexes, as well as the flagellar system are also expressed to higher levels in psyllids. We also analyzed the relative expression levels of Sec-delivered effectors, which are considered key virulence factors of Las. This work advances our understanding of the HLB biology and offers novel insight into the HLB pathogenesis.
Project description:Citrus Huanglongbing (HLB, or greening) is one of the most severe diseases of citrus. Plant disease symptom development is considered to be the consequence of a number of molecular, cellular and physiological changes, and may also be associated with host defense responses. Understanding citrus host response to HLB may contribute to the development of new strategies to control this destructive disease. We performed microarray analysis to identify the differentially expressed genes in sweet orange in response to HLB infection using the Affymetrix GeneChipM-BM-. citrus genome array. Two-year-old seedlings of M-bM-^@M-^XMadam VinousM-bM-^@M-^Y sweet orange (Citrus sinensis L. Osbeck) were inoculated by grafting with bud sticks from HLB-diseased, PCR positive sweet orange plants. For mock-inoculated controls, the same types of plants were grafted with bud sticks from HLB-free, PCR negative sweet orange. At 7 months after inoculation, mature leaves were sampled from 3 individual HLB-diseased plants, and healthy leaves from 3 mock-inoculated plants as control. Total RNA was extracted from leaf samples and hybridized on Affymetrix microarrays.
Project description:Candidatus Liberibacter asiaticus (Las), a non-culturable phloem-limited bacterium, is the suspected causal agent of Huanglongbing (HLB) in Florida. HLB is one of the most devastating diseases of citrus and no resistant cultivars have been identified to date, though tolerance has been suggested in the genus Poncirus. A recent study conducted in our laboratory demonstrated tolerance of US-897, a hybrid of Poncirus trifoliata (L. Raf.) and the susceptible ‘Cleopatra’ mandarin (Citrus reticulata Blanco), to Ca. L. asiaticus, the presumed causal agent of HLB in Florida (Albrecht & Bowman, HortScience 46 (2011) 16-22). This study compares transcriptional changes in tolerant US-897 and susceptible ‘Cleopatra’ mandarin seedlings in response to infection with Las using the Affymetrix GeneChip citrus array with the main objective of identifying genes associated with tolerance to HLB. Such genes may be suitable as potential targets for biotechnology approaches, providing one strategy to possibly control this destructive disease of citrus.
Project description:Huanglongbing (HLB) (=citrus greening) is a destructive disease of citrus which is caused by a fastidious, phloem-inhabiting bacterium of the genus Candidatus Liberibacter. Large-scale analysis of gene expression changes in ‘Valencia’ orange leaves were studied during the course of 19 weeks after inoculation with Ca. L. asiaticus using the Affymetrix GeneChip® citrus genome array to provide new insights into the molecular basis of citrus response to this pathogen. Of the more than 33,000 probe sets on the microarray 21,067 were expressed in the leaves, of which 279 and 515 were differentially expressed (FDR ≤ 0.05) five to nine and 13-17 weeks after inoculation, respectively. Results from semi-quantitative RT-PCR analysis performed on 14 selected genes were highly correlated with those observed with the microarray. Gene expression changes involved a variety of different processes including cell defense, transport, cellular organization, photosynthesis, and carbohydrate metabolism. Notable was the pathogen-induced accumulation of transcripts for a phloem-specific lectin PP2-like protein. Transcriptional changes and their relation to disease symptom development are discussed. This is the first study of transcriptional profiling in citrus in response to liberibacter infection using microarray technology. Huanglongbing (HLB) is a destructive disease of citrus which is suspected to be caused by a phloem-inhabiting bacterium of the genus Candidatus Liberibacter. Large-scale analysis of gene expression changes in ‘Valencia’ orange (C. sinensis) leaves were studied during the course of 19 weeks after inoculation with Ca. L. asiaticus (Las), the pathogen associated with HLB in Florida, using the Affymetrix GeneChip® citrus genome array to provide new insights into the molecular basis of citrus response to this pathogen.
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.
Project description:Candidatus Liberibacter asiaticus (Las), a non-culturable phloem-limited bacterium, is the suspected causal agent of Huanglongbing (HLB) in Florida. HLB is one of the most devastating diseases of citrus and no resistant cultivars have been identified to date, though tolerance has been suggested in the genus Poncirus. A recent study conducted in our laboratory demonstrated tolerance of US-897, a hybrid of Poncirus trifoliata (L. Raf.) and the susceptible ‘Cleopatra’ mandarin (Citrus reticulata Blanco), to Ca. L. asiaticus, the presumed causal agent of HLB in Florida (Albrecht & Bowman, HortScience 46 (2011) 16-22). This study compares transcriptional changes in tolerant US-897 and susceptible ‘Cleopatra’ mandarin seedlings in response to infection with Las using the Affymetrix GeneChip citrus array with the main objective of identifying genes associated with tolerance to HLB. Such genes may be suitable as potential targets for biotechnology approaches, providing one strategy to possibly control this destructive disease of citrus. Fifteen months-old seedlings of the genotypes 'Cleopatra' mandarin and US-897 were graft-inoculated with non-infected (control) or Las-infected tissue from greenhouse-grown 'Valencia' (C. sinensis L.) plants. Six non-infected control plants each from 'Cleopatra' (MC) and US-897 (TC) and six infected plants each from Cleopatra (MI) and US-897 (TI) were used. Four to six leaves per plant were excised at 32 weeks after inoculation (wai) and immediately frozen in liquid nitrogen. Total RNA was extracted from all 24 plants. Equal amounts of RNA from two samples were pooled to obtain three biological replicates per genotype and treatment and used for hybridization on Affymetrix citrus microarrays.