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Citrus Vascular Proteomics Highlights the Role of Peroxidases and Serine Proteases during Huanglongbing Disease Progression.

ABSTRACT: Huanglongbing (HLB) is the most devastating and widespread citrus disease. All commercial citrus varieties are susceptible to the HLB-associated bacterium, Candidatus Liberibacter asiaticus (CLas), which resides in the phloem. The phloem is part of the plant vascular system and is involved in sugar transport. To investigate the plant response to CLas, we enriched for proteins surrounding the phloem in an HLB susceptible sweet orange variety, Washington navel (Citrus sinensis (L) Osbeck). Quantitative proteomics revealed global changes in the citrus proteome after CLas inoculation. Plant metabolism and translation were suppressed, whereas defense-related proteins such as peroxidases, proteases and protease inhibitors were induced in the vasculature. Transcript accumulation and enzymatic activity of plant peroxidases in CLas infected sweet orange varieties under greenhouse and field conditions were assessed. Although peroxidase transcript accumulation was induced in CLas infected sweet orange varieties, peroxidase enzymatic activity varied. Specific serine proteases were up-regulated in Washington navel in the presence of CLas based on quantitative proteomics. Subsequent activity-based protein profiling revealed increased activity of two serine proteases, and reduced activity of one protease in two C. sinensis sweet orange varieties under greenhouse and field conditions. The observations in the current study highlight global reprogramming of the citrus vascular proteome and differential regulation of enzyme classes in response to CLas infection. These results open an avenue for further investigation of diverse responses to HLB across different environmental conditions and citrus genotypes.

SUBMITTER: Franco JY

PROVIDER: S-EPMC7710146 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Huanglongbing (HLB or Citrus Greening Disease) is a destructive citrus disease that has impacted the Florida citrus industry as well as global citrus industry for many years. There is yet to be a practical cure for HLB. Insecticide use targeting the psyllid vector and the burning, destruction and removal of all infected plants are the best solutions as of now. However, this is only a temporary and costly fix. The damage caused by HLB itself, the destruction of infected trees, and indirect effects in other economic sectors costs the Florida economy approximately $900 million per year. There are also problems with insecticide use; the high dosage needed the adequately suppress the transmission of the disease would contaminate the environment, rendering this solution unsustainable. Alternative solutions are needed. In the hopes of uncovering an alternative solution, we investigated the protein and phosphoprotein differences between healthy and diseased citrus plants to identify potential biomarkers essential towards fighting HLB within the trees themselves. For our experimental procedure, we grinded 2 grams of fresh leaf weight from the species Citrus sinensis (sweet orange). Phenol extraction was used to extract the proteins from four biological replicates of the control (healthy) leaves and four biological replicates of the treatment (diseased) leaves. Utilizing a Bradford assay and a 1-D gel electrophoresis, we evaluated the quality and quantity of the samples. Equal amounts of protein from the eight samples were digested with trypsin for 16 hours. Afterwards, we split each sample into two groups: one for phospho-enrichment and the other for total analysis. Phosphopeptides were enriched with the NuTip enrichment procedure for analysis of the phosphopeptides. Total peptides were cleaned up using ZipTip solid phase extraction. We ran the phosphopeptides and total peptides on an LC-MS/MS system, and the raw data files were processed with the Proteome Discoverer software using the citrus database downloaded from NCBI.

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Citrus Vascular Proteomics Highlights the Role of Peroxidases and Serine Proteases during Huanglongbing Disease Progression.

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