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RNA-Seq analysis of Citrus reticulata in the early stages of Xylella fastidiosa infection reveals auxin-related genes as a defense response.

Rodrigues CM, de Souza AA, Takita MA, Kishi LT, Machado MA - BMC Genomics (2013)

Bottom Line: A set of genes considered key elements in the resistance was used to confirm its regulation in mandarin compared with the susceptible sweet orange.Gene expression analysis of mock inoculated and infected tissues of Ponkan mandarin identified 667 transcripts repressed and 724 significantly induced in the later.We also hypothesized the induction of auxin-related genes indicates that resistant plants initially recognize X. fastidiosa as a necrotrophic pathogen.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Biotecnologia, Centro APTA Citros Sylvio Moreira, CP4, Cordeirópolis, SP 13490-970, Brazil. marcos@centrodecitricultura.br.

ABSTRACT

Background: Citrus variegated chlorosis (CVC), caused by Xylella fastidiosa, is one the most important citrus diseases, and affects all varieties of sweet orange (Citrus sinensis L. Osb). On the other hand, among the Citrus genus there are different sources of resistance against X. fastidiosa. For these species identifying these defense genes could be an important step towards obtaining sweet orange resistant varieties through breeding or genetic engineering. To assess these genes we made use of mandarin (C. reticulata Blanco) that is known to be resistant to CVC and shares agronomical characteristics with sweet orange. Thus, we investigated the gene expression in Ponkan mandarin at one day after infection with X. fastidiosa, using RNA-seq. A set of genes considered key elements in the resistance was used to confirm its regulation in mandarin compared with the susceptible sweet orange.

Results: Gene expression analysis of mock inoculated and infected tissues of Ponkan mandarin identified 667 transcripts repressed and 724 significantly induced in the later. Among the induced transcripts, we identified genes encoding proteins similar to Pattern Recognition Receptors. Furthermore, many genes involved in secondary metabolism, biosynthesis and cell wall modification were upregulated as well as in synthesis of abscisic acid, jasmonic acid and auxin.

Conclusions: This work demonstrated that the defense response to the perception of bacteria involves cell wall modification and activation of hormone pathways, which probably lead to the induction of other defense-related genes. We also hypothesized the induction of auxin-related genes indicates that resistant plants initially recognize X. fastidiosa as a necrotrophic pathogen.

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Relative quantification of genes related auxin pathway in Ponkan mandarin and sweet orange by RT-qPCR. cDNA samples were prepared using RNA from xylem tissue from Ponkan mandarin and sweet orange, after 1 day of infection with X. fastidiosa or not (control) (three biological replicates). The bars indicate the standard deviation of the means. (*) indicates significant difference (P ≥ 0.05) between the mean values obtained for each gene compared with the control.
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Figure 5: Relative quantification of genes related auxin pathway in Ponkan mandarin and sweet orange by RT-qPCR. cDNA samples were prepared using RNA from xylem tissue from Ponkan mandarin and sweet orange, after 1 day of infection with X. fastidiosa or not (control) (three biological replicates). The bars indicate the standard deviation of the means. (*) indicates significant difference (P ≥ 0.05) between the mean values obtained for each gene compared with the control.

Mentions: To confirm that auxin signaling related genes are indeed upregulated only in Ponkan mandarin in response to X. fastidiosa infection we also evaluated the expression of auxin marker-genes by RT-qPCR in Pera sweet orange susceptible variety and in Ponkan mandarin at one day after X. fastidiosa infection. As shown in Figure 5, all auxin related-genes were significantly induced only in Ponkan mandarin. In Pera sweet orange, the genes were significantly repressed (E3 and ARF19) or showed no significant change (IAA9, TIR1 and BIG). This result evidences that auxin is induced as a resistance response against X. fastidiosa during the early stage of infection. Recognition of PAMPs or DAMPs that somehow resemble necrotrophic pathogens may mediate this response. However, this recognition occurs mainly during the early stage of infection since we observed a gradual decrease in expression of auxin related-genes along the time course of infection (Figure 6). After 21 days, no auxin related-gene was expressed, whereas expression of salicylic acid (SA) marker-gene increased (Figure 6). This result agrees with De Souza et al. [6,7] where an upregulation of SA related-genes was observed in Ponkan mandarin at 30 days after X. fastidiosa inoculation. After this time point, the bacterial population decreases to a point where it could not be isolated [3]. These results suggest that the resistant plant changes its mechanism of defense during X. fastidiosa infection: the initial response involves the participation of auxin while later on SA becomes important. It is to note that the change occurs approximately at the time when X. fastidiosa forms a structured biofilm. In this growth condition this bacterium expresses specific genes and proteins necessary for its adaptation and pathogenicity in the host [3,4,48,49]. Therefore other proteins expressed in biofilm condition could be later recognized by the plant. Nevertheless, how the resistant plant indeed recognizes X. fastidiosa to trigger different pathways in the resistance response remains to be discovered. Other downstream defense-genes upregulated in Ponkan mandarin after X. fastidiosa infection are represented at the Table 2. These genes might contribute to increase the resistance response in Ponkan mandarin to X. fastidiosa culminating in its elimination in the plant.


RNA-Seq analysis of Citrus reticulata in the early stages of Xylella fastidiosa infection reveals auxin-related genes as a defense response.

Rodrigues CM, de Souza AA, Takita MA, Kishi LT, Machado MA - BMC Genomics (2013)

Relative quantification of genes related auxin pathway in Ponkan mandarin and sweet orange by RT-qPCR. cDNA samples were prepared using RNA from xylem tissue from Ponkan mandarin and sweet orange, after 1 day of infection with X. fastidiosa or not (control) (three biological replicates). The bars indicate the standard deviation of the means. (*) indicates significant difference (P ≥ 0.05) between the mean values obtained for each gene compared with the control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852278&req=5

Figure 5: Relative quantification of genes related auxin pathway in Ponkan mandarin and sweet orange by RT-qPCR. cDNA samples were prepared using RNA from xylem tissue from Ponkan mandarin and sweet orange, after 1 day of infection with X. fastidiosa or not (control) (three biological replicates). The bars indicate the standard deviation of the means. (*) indicates significant difference (P ≥ 0.05) between the mean values obtained for each gene compared with the control.
Mentions: To confirm that auxin signaling related genes are indeed upregulated only in Ponkan mandarin in response to X. fastidiosa infection we also evaluated the expression of auxin marker-genes by RT-qPCR in Pera sweet orange susceptible variety and in Ponkan mandarin at one day after X. fastidiosa infection. As shown in Figure 5, all auxin related-genes were significantly induced only in Ponkan mandarin. In Pera sweet orange, the genes were significantly repressed (E3 and ARF19) or showed no significant change (IAA9, TIR1 and BIG). This result evidences that auxin is induced as a resistance response against X. fastidiosa during the early stage of infection. Recognition of PAMPs or DAMPs that somehow resemble necrotrophic pathogens may mediate this response. However, this recognition occurs mainly during the early stage of infection since we observed a gradual decrease in expression of auxin related-genes along the time course of infection (Figure 6). After 21 days, no auxin related-gene was expressed, whereas expression of salicylic acid (SA) marker-gene increased (Figure 6). This result agrees with De Souza et al. [6,7] where an upregulation of SA related-genes was observed in Ponkan mandarin at 30 days after X. fastidiosa inoculation. After this time point, the bacterial population decreases to a point where it could not be isolated [3]. These results suggest that the resistant plant changes its mechanism of defense during X. fastidiosa infection: the initial response involves the participation of auxin while later on SA becomes important. It is to note that the change occurs approximately at the time when X. fastidiosa forms a structured biofilm. In this growth condition this bacterium expresses specific genes and proteins necessary for its adaptation and pathogenicity in the host [3,4,48,49]. Therefore other proteins expressed in biofilm condition could be later recognized by the plant. Nevertheless, how the resistant plant indeed recognizes X. fastidiosa to trigger different pathways in the resistance response remains to be discovered. Other downstream defense-genes upregulated in Ponkan mandarin after X. fastidiosa infection are represented at the Table 2. These genes might contribute to increase the resistance response in Ponkan mandarin to X. fastidiosa culminating in its elimination in the plant.

Bottom Line: A set of genes considered key elements in the resistance was used to confirm its regulation in mandarin compared with the susceptible sweet orange.Gene expression analysis of mock inoculated and infected tissues of Ponkan mandarin identified 667 transcripts repressed and 724 significantly induced in the later.We also hypothesized the induction of auxin-related genes indicates that resistant plants initially recognize X. fastidiosa as a necrotrophic pathogen.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Biotecnologia, Centro APTA Citros Sylvio Moreira, CP4, Cordeirópolis, SP 13490-970, Brazil. marcos@centrodecitricultura.br.

ABSTRACT

Background: Citrus variegated chlorosis (CVC), caused by Xylella fastidiosa, is one the most important citrus diseases, and affects all varieties of sweet orange (Citrus sinensis L. Osb). On the other hand, among the Citrus genus there are different sources of resistance against X. fastidiosa. For these species identifying these defense genes could be an important step towards obtaining sweet orange resistant varieties through breeding or genetic engineering. To assess these genes we made use of mandarin (C. reticulata Blanco) that is known to be resistant to CVC and shares agronomical characteristics with sweet orange. Thus, we investigated the gene expression in Ponkan mandarin at one day after infection with X. fastidiosa, using RNA-seq. A set of genes considered key elements in the resistance was used to confirm its regulation in mandarin compared with the susceptible sweet orange.

Results: Gene expression analysis of mock inoculated and infected tissues of Ponkan mandarin identified 667 transcripts repressed and 724 significantly induced in the later. Among the induced transcripts, we identified genes encoding proteins similar to Pattern Recognition Receptors. Furthermore, many genes involved in secondary metabolism, biosynthesis and cell wall modification were upregulated as well as in synthesis of abscisic acid, jasmonic acid and auxin.

Conclusions: This work demonstrated that the defense response to the perception of bacteria involves cell wall modification and activation of hormone pathways, which probably lead to the induction of other defense-related genes. We also hypothesized the induction of auxin-related genes indicates that resistant plants initially recognize X. fastidiosa as a necrotrophic pathogen.

Show MeSH
Related in: MedlinePlus