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RNA-SEQ ANALYSIS Of Vitis vinifera DISPLAYING SYMPTOMS OF PIERCE'S RNA-SEQ ANALYSIS Of Vitis vinifera DISPLAYING SYMPTOMS OF PIERCE'S DISEASE,EXPERIMENTAL INFECTION WITH THE PHYTOBACTERIUM Xylella DISEASE,EXPERIMENTAL INFECTION WITH THE PHYTOBACTERIUM Xylella
fastidiosafastidiosa
Menegidio, FB¹; Santos, DS¹; Alencar, VC¹; Costa de Oliveira, RL¹; Almeida, RP²; Nunes, LR³¹Núcleo Integrado de Biotecnologia, Universidade de Mogi das Cruzes, Brazil
²Department of Environmental Science, Policy and Management, College of Natural Resources, University of California at Berkeley, Berkeley, CA, USA
³Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, BrazilCorresponding authors: [email protected]
Pierce's disease (PD) is a highly destructive disease, responsible for great losses in the North American viticulture industry. It is caused by the xylem-limited bacterium Xylella fastidiosa and characterized by blockage of xylem vessels by bacterial aggregates, which leads to water stress and chlorosis of leaf tissue, among other symptoms, affecting fruit development and overall plant productivity. However, the exact mechanisms that lead to PD development in vineyards are still poorly understood, which prompted us to employ RNA-seq analysis, in an attempt to perform a global-scale evaluation of the main metabolic alterations that occur in PD-affected plants. Thus, we inoculated four 3-month-old Vitis vinifera plants (Thompson Seedless variety) with Xylella fastidiosa (Temecula strain), which were next incubated in a greenhouse for four months. At this point, PD symptoms were clearly visible and RNA from affected leaves was extracted (Fig. 1). RNA was also extracted from the leaves of four healthy, uninfected plants that were incubated under the same conditions, to serve as control. These RNA samples were then sequenced in an Illumina platform and transcripts were mapped against the entire Vitis vinifera reference genome, with the aid of Tophat (Tab. 1). After mapping, the relative abundance of transcripts was calculated with Cufflinks and the differentially expressed genes were identified with Cuffdiff (Fig. 2). A total of 5,779 differentially expressed transcripts were obtained after this analysis, identifying several pathways that seem to be affected in diseased plants, such as photosynthesis, wax production and synthesis of chalcones, among others (Fig. 3).
Figure 3. A comparative PageMan display of modulated pathways in vine library uninfected and infected with Xylella fastidiosa Temecula. The fold changes of gene expression levels were input into PageMan and subjected to a Wilcoxon test. Pathways that were significantly up-regulated are colored in blue, and those colored in red were significantly down-regulated. The names of pathways of interest are indicated on the right panel.
Figure 1. The leaves of plants showing symptoms of Pierce's Disease. The photos were taken of four plants Thompson Seedless (Vitis vinifera L.) with obvious symptoms of Pierce's disease after 4 months of experimental infection by X. fastidiosa Temecula. Petioles were harvested and macerated for RNA extraction and analysis of gene expression by RNA-Seq. TP1 (A), TP2 (B), TP3 (C) and TP4 (D).
Figure 2. Tuxedo suite for RNA-seq differential expression analysis. Pre-processed reads from two groups are mapped by TopHat. The resulting files are used for transcript assembly by Cufflinks with a given *.gtf file. Cuffdiff performs statistical testinge to identify differential expression. In addition, the differentially expressed genes were also functionally analyzed using the MapMan software.
PAGEMAN MAPMANMERCARTOR(Annotation)
CUFFDIFF
CUFFLINKSTOPHATFASTQ
GROOMER
FASTQQUALITYTRIMMER
CLIPADAPTER
RNA-SEQREADS
RESULTS
Table 1. Number of sequences obtained for each vine library uninfected (HP1, HP2, HP3, HP4) and infected with Xylella fastidiosa Temecula (TP1, TP2, TP3, TP4).
Reads Input Reads Mapped Overall read alignment rateHP1 40826665 27343135 67.0%
HP2 25865100 18532857 71.7%
HP3 18338120 6705794 36.6%
HP4 24715432 18621923 75.3%
TP1 27621134 19696636 71.3%
TP2 25455453 17835909 70.1%
TP3 19161712 7307361 38.1%
TP4 26747247 18745448 70.1%
