TARGETING VITAL GENES OF PHYTOPATHOGENIC FUNGUS MARSSONINA CORONARIA USING RNAi APPROACH FOR HOST INDUCED GENE SILENCING IN APPLE
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Date
2018
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UHF,NAUNI
Abstract
ABSTRACT
Marssonina coronaria (Ellis & J.J. Davis) is economically very important apple pathogen fungus causing
severe premature defoliation in apple. All the available commercial cultivars of apple are susceptible to this pathogen.
The apple orchard survey revealed that the disease was widespread in three districts of HP. Despite this, nothing much
has been studied about the molecular biology of this fungus and its genome remains unannotated. A highly virulent
strain of M. coronaria was isolated and characterized. Among different media tested, maximum colony diameter was
produced on PPDA whereas, maximum fungal dry weight and conidia were produced in PPDB grown cultures at
25°C. Treatment of conidia with EGTA and anti-calmodulin drug Trifluoperazine dihydrochloride in PPDB resulted
in the inhibition of spore germination as compared to the control. Eight RNA extraction methods were evaluated to
yield high-quality and melanin-free RNA. Age of fungus culture on solid and liquid medium affected the total RNA
yield, and melanin contamination. TRIzol™ Reagent+RNA Clean & Concentrator™-5 and Ambion-PureLink® RNA
Mini Kit were found to be the best methods which were applied for RT-PCR, subsequent PCR amplification, and
isolation of calmodulin, β-tubulin, HSP-90 co-chaperone Cdc37, SNF2 family domain, C6 zinc finger domain and
transcription activator gene sequences from M. coronaria. Semi-qRT-PCR of β-tubulin gene revealed its suitability as
reference housekeeping gene for gene expression studies and explored for molecular detection of M. coronaria from
infected apple tissues. Two essential genes calmodulin and HSP-90 were opted for dsRNA synthesis using pL4440
vector and development of hairpin-RNAi constructs using pSilent-1 and pCAMBIA-1300 vectors. Treatment of
conidia suspension with 10 μL of dsRNA (750 ng/ul) targeting CALM and HSP-90 gene resulted in 11 and 34
colonies, respectively which were significantly less than that of the water control (74). These treated conidia as well as
the topical application of dsRNA resulted in reduced symptom appearance on leaf surface of apple varieties. ATMT
was standardized with mycelia plugs and fragmented mycelia using pCAMBIA-1304 with parameters viz.,
Agrobacterium concentration (OD600 - 0.5), AS (250μM), coculture time (48 h), and temperature (24°C) and
membrane (Whatman filter paper). pCAMBIA-1304 transformants exhibited strong GUS staining, GFP fluorescence
and hygromycin resistance even after five successive culturing, and were PCR positive for the hpt and gus genes.
Standardized ATMT was employed to generate silenced mutants with CALM and HSP-90 hairpin constructs and
confirmed by PCR for hpt gene. Also, qRT-PCR analysis showed drastic reduction in the transcript level of these
genes in mutants as compared to control fungi. The mutants also showed significant reduction in growth, sporulation,
mycelia dry weight and pigmentation. In vitro detached apple leaf infection revealed that the spores of the silenced
mutants of HSP-90 target gene showed low virulence with delayed symptom appearance whereas, the CALM mutants
exhibited loss of virulence. To conclude, we have elucidated the role of calmodulin and HSP-90 co-chaperone Cdc37
genes of M. coronaria by utilizing the RNAi approach. Further, this study will widen the molecular biological
knowledge of this fungus and host pathogen interaction as well as HIGS to control apple leaf blotch.
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