Characterization of secreted in Xylem (SIX) genes during plant- pathogen interaction of Fusarium oxysporum f.sp. cubense (Foc), Foc1 and Foc4 in Musa acuminata cv. ‘Berangan’ / Kausalyaa Kaliapan

Kausalyaa , Kaliapan (2024) Characterization of secreted in Xylem (SIX) genes during plant- pathogen interaction of Fusarium oxysporum f.sp. cubense (Foc), Foc1 and Foc4 in Musa acuminata cv. ‘Berangan’ / Kausalyaa Kaliapan. Masters thesis, Universiti Malaya.

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Abstract

Fusarium wilt of banana is a lethal disease caused by Fusarium oxysporum f.sp. cubense (Foc), a soil-borne pathogen that curbs the production of this crop. Small effector proteins known as Secreted in Xylem (SIX), are secreted into the xylem sap of the host plants triggering virulence and interferes with the defence responses of the host plants. Knowledge and comprehension of the interaction between Foc and banana plants during the infection is essential to expand methods to mitigate the disease’s damage. Therefore, studies on SIX genes are crucial as they have significant impact on Foc’s pathogenicity and play substantial role in the development of molecular framework and disease control approaches for banana. In this study, the responsible SIX genes for this pathogenic reaction were identified, characterized and their gene expression patterns were analysed during the infection. A comparative whole genome analysis was also carried out between Malaysian Foc1_C2HIR and Foc4_C1HIR isolates. A total of 48 cv. ‘Berangan’ plantlets were used where 32 plantlets were subjected to Foc infection (Foc1 and Foc4) while 16 plantlets were used as controls (without infection). Collection of plant samples were made at four different time points, 0 day, 2 weeks, 4 weeks and 5 weeks for molecular studies while the internal and external symptoms were confirmed by visual assessment and scored based on the percentage of infection on the 5th week post-inoculation. Following the data analysis, the ‘Berangan’ cultivar was confirmed to be highly susceptible to Foc infection. The Rhizome Discoloration Index (RDI) and Leaf Symptom Index (LSI) of Foc4-inoculated plants (7.25; 4.25 respectively) were higher compared to Foc1- inoculated plants (5.0; 3.25 respectively) which suggested that Foc4 is more virulent than Foc1 in cv. Berangan. Seven SIX genes i.e. SIX1, SIX2, SIX4, SIX6, SIX8a, SIX9a, SIX13 were identified to be present in Foc4 DNA, while only SIX9b was identified in Foc1 DNA and validated via sequencing analysis. Five of the SIX genes were shortlisted to be further studied on their expression pattern during infection by performing real-time PCR (qPCR) on fungal RNA from infected banana root samples. TEF1α and TUB were selected as the endogenous genes for normalisation. In the gene expression analysis conducted on Foc1 and Foc4 infected plants, two different patterns of expression were observed. In Foc4- inoculated plants, SIX1, SIX6, SIX8a and SIX9a were significantly upregulated on the 2nd and 5th week, whereas in Foc1-inoculated plants, significant upregulation of SIX9b was observed at a later stage which was on the 5th week. Besides, whole genome sequencing analysis for both Foc1_C2HIR and Foc4_C1HIR Malaysian isolates revealed that they had a respective genome size of 53 Mb and 55 Mb. Further analysis also revealed the genome features, SIX gene structures, putative virulence-associated genes, and functional groups of proteins identified in C2HIR and C1HIR isolates. These findings will aid in understanding the mechanism of potential disease control targets in these plants, including the improvement of diagnostics and breeding programmes.

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