Deciphering the mode of action of a mutant Allium sativum Leaf Agglutinin (mASAL), a potent antifungal protein on Rhizoctonia solani.
BACKGROUND
Mutant Allium sativum leaf agglutinin (mASAL) is a potent, biosafe, antifungal protein that exhibits fungicidal activity against different phytopathogenic fungi, including Rhizoctonia solani.


METHODS
The effect of mASAL on the morphology of R.solani was monitored primarily by scanning electron and light microscopic techniques. Besides different fluorescent probes were used for monitoring various intracellular changes associated with mASAL treatment like change in mitochondrial membrane potential (MMP), intracellular accumulation of reactive oxygen species (ROS) and induction of programmed cell death (PCD). In addition ligand blot followed by LC-MS/MS analyses were performed to detect the putative interactors of mASAL.


RESULTS
Knowledge on the mode of function for any new protein is a prerequisite for its biotechnological application. Detailed morphological analysis of mASAL treated R. solani hyphae using different microscopic techniques revealed a detrimental effect of mASAL on both the cell wall and the plasma membrane. Moreover, exposure to mASAL caused the loss of mitochondrial membrane potential (MMP) and the subsequent intracellular accumulation of reactive oxygen species (ROS) in the target organism. In conjunction with this observation, evidence of the induction of programmed cell death (PCD) was also noted in the mASAL treated R. solani hyphae. Furthermore, we investigated its interacting partners from R. solani. Using ligand blots followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses, we identified different binding partners including Actin, HSP70, ATPase and 14-3-3 protein.


CONCLUSIONS
Taken together, the present study provides insight into the probable mode of action of the antifungal protein, mASAL on R. solani which could be exploited in future biotechnological applications.
Division of Plant Biology, Bose Institute, Centenary Campus, P1/12, CIT Scheme, VIIM, Kankurgachi, Kolkata, 700054, West Bengal, India. prithwi11@gmail.com.
Prithwi
P
Ghosh
Division of Plant Biology, Bose Institute, Centenary Campus, P1/12, CIT Scheme, VIIM, Kankurgachi, Kolkata, 700054, West Bengal, India. amitroy_81@yahoo.co.in.|Present address: Chemical Ecology, Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Alnarp, SE-230 53, Sweden. amitroy_81@yahoo.co.in.
Amit
A
Roy
The Protein Analysis Facility, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland. dhess@fmi.ch.
Daniel
D
Hess
Division of Plant Biology, Bose Institute, Centenary Campus, P1/12, CIT Scheme, VIIM, Kankurgachi, Kolkata, 700054, West Bengal, India. ghosh.anupama@jcbose.ac.in.
Anupama
A
Ghosh
Division of Plant Biology, Bose Institute, Centenary Campus, P1/12, CIT Scheme, VIIM, Kankurgachi, Kolkata, 700054, West Bengal, India. sampa@jcbose.ac.in.
Sampa
S
Das
D000373:Agglutinins / Q000302:isolation & purification / Q000494:pharmacology*; D000935:Antifungal Agents / Q000302:isolation & purification / Q000494:pharmacology*; D017209:Apoptosis; D002462:Cell Membrane / Q000187:drug effects; D002473:Cell Wall / Q000187:drug effects; D002853:Chromatography, Liquid; D005737:Garlic / Q000737:chemistry*; D025301:Hyphae / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D050296:Microbial Viability / Q000187:drug effects; D008853:Microscopy; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000502:physiology; D050505:Mutant Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D025941:Protein Interaction Mapping; D017382:Reactive Oxygen Species / Q000032:analysis; D012232:Rhizoctonia / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D053719:Tandem Mass Spectrometry
D000373:Agglutinins; D000935:Antifungal Agents; D050505:Mutant Proteins; D017382:Reactive Oxygen Species