Comparative analysis of the transcriptomes of the calyx abscission zone of sweet orange insights into the huanglongbing-associated fruit abscission

Authors: Zhao, Wei; Baldwin, Elizabeth - Liz; Bai, Jinhe; Plotto, Anne; IREY, MIKE - Southern Gardens Citrus

Submitted to: Horticulture Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/10/2019
Publication Date: 6/1/2019
Citation: Zhao, W., Baldwin, E.A., Bai, J., Plotto, A., Irey, M. Comparative analysis of the transcriptomes of the calyx abscission zone of sweet orange insights in the huanglongbing-associated fruit abscission. Horticulture Research. 6:71. 2019. https://doi.org/10.1038/s41438-019-0152-4.
DOI: https://doi.org/10.1038/s41438-019-0152-4

Interpretive Summary: Citrus greening or huanglongbing (HLB) disease is caused by a bacteria that is transmitted to citrus tree leaves by a small insect. This disease is associated with an increase in pre-harvest fruit drop which has drastically reduced yields and for which the molecular mechanisms remain unknown. Recent studies have implicated a secondary infection by fungus in enhancing fruit drop from HLB-affected trees. In order to understand the molecular basis of the HLB-associated fruit abscission, genetic studies of the stems where the fruit separates (abscises) from the tree (abscission zone) were analyzed and compared among healthy fruit from healthy trees, and fruit dropped or retained from HLB-affected trees upon shaking the trees, by means of RNA-Sequencing analysis (RNA-Seq). This type of study shows which genes are turned on or off, and results showed that genes that induce the abscission hormone ethylene and response to fungi were turned on in fruit abscission zones of dropped fruit. This confirms that the fruit drop is likely exacerbated by a secondary fungal infection which promotes the plant hormone ethylene to be produced, which in turn, promotes abscission or fruit drop.

Technical Abstract: Citrus greening or huanglongbing (HLB) disease is caused by Candidatus liberibacter asiaticus (CLas) and associated with an increase in pre-harvest fruit drop, for which the molecular mechanisms remain unknown. Recent studies have implicated a secondary infection by fungus Lasiodiplodia theobromae in enhancing fruit drop from HLB-affected trees. In order to understand the molecular basis of the HLB-associated fruit abscission, transcriptomes in citrus calyx abscission zones were analyzed and compared among healthy fruit (H) from healthy (CLas negative) trees, and fruit dropped (D) or retained (R) from HLB-affected (CLas positive) trees upon shaking the trees, by means of RNA-Sequencing analysis (RNA-Seq). Comparison of the transcriptomic profiles revealed 1209, 1739 and 927 significantly (p<0.05) differentially expressed genes (DEGs) between D and R, D and H, R and H, respectively. Wilcoxon test of the whole dataset of DEGs revealed that consistently up-regulated genes in D versus R and H fruit included genes related to biotic stress, lipid and secondary metabolism, whereas genes related to carbohydrate metabolism and cell wall were down-regulated. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs indicated that the most significant GO term was “response to chitin” (p= 9.70E-09), while “response to jasmonic acid” and “phenylpropanoid biosynthesis” were also significant. Among KEGG pathways, “alpha-linolenic acid metabolism” was the most significant (p < 0.005), which produces jasmonates (JA). In addition, “plant hormone signal transduction” and “phenylpropanoid biosynthesis” were also significant pathways (p < 0.05). Hierarchical clustering analysis indicated genes related to synthesis and signaling of ethylene (ET) and JA were consistently up-regulated, while abscisic acid, auxin, brassinosteroid, cytokinin, and gibberellin were generally down-regulated in D versus R and H fruit. And in agreement with the hormone expression profiles, substantial numbers of downstream JA/ET-responsive defense (the hallmark of anti-fungal defense) related genes were up-regulated in D fruit as well. Quantitative RT-PCR results confirmed RNA-Seq results for 30 representative genes covering each of the DEG categories. In conclusion, HLB-associated fruit abscission is mediated by JA/ET signaling, likely in response to a secondary fungal infection.