doi: 10.1038/s41598-017-17248-7.
Biotrophy-necrotrophy switch in pathogen evoke differential response in resistant and susceptible sesame involving multiple signaling pathways at different phases
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- PMID: 29222513
- PMCID: PMC5722813
- DOI: 10.1038/s41598-017-17248-7
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Biotrophy-necrotrophy switch in pathogen evoke differential response in resistant and susceptible sesame involving multiple signaling pathways at different phases
Sci Rep.
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Abstract
Infection stages of charcoal rot fungus Macrophomina phaseolina in sesame revealed for the first time a transition from biotrophy via BNS (biotrophy-to-necrotrophy switch) to necrotrophy as confirmed by transcriptional studies. Microscopy using normal and GFP-expressing pathogen showed typical constricted thick intercellular bitrophic hyphae which gave rise to thin intracellular necrotrophic hyphae during BNS and this stage was delayed in a resistant host. Results also show that as the pathogen switched its strategy of infection, the host tailored its defense strategy to meet the changing situation. Less ROS accumulation, upregulation of ROS signaling genes and higher antioxidant enzyme activities post BNS resulted in resistance. There was greater accumulation of secondary metabolites and upregulation of secondary metabolite-related genes after BNS. A total of twenty genes functioning in different aspects of plant defense that were monitored over a time course during the changing infection phases showed a coordinated response. Experiments using phytohormone priming and phytohormone inhibitors showed that resistance resulted from activation of JA-ET signaling pathway. Most importantly this defense response was more prompt in the resistant than the susceptible host indicating that a resistant host makes different choices from a susceptible host during infection which ultimately influences the severity of the disease.
Conflict of interest statement
The authors declare that they have no competing interests.
Figures
Macrophomina phaseolina infection of sesame shows a hemibiotrophic life cycle during infection of resistant (Nirmala) and susceptible (VRI-1) varieties of sesame. (a,c) Formation of intracellular vesicles in roots (3 hpi). (b,d) Hyphopodia arising from vegetative hyphae on sesame root surface during early biotrophic phase (3–6 hpi). (e,g) Confocal microscopy showing GFP-expressing runner hyphae on root surface of resistant variety Nirmala and susceptible VRI-1 (9–12 hpi). (f,h,i,k) confocal microscopy showing presence of typical constricted biotrophic hyphae within root tissues of both varieties (12 hpi). (j,l) GFP expressing intercellular biotrophic hyphae (16–24 hpi). (m,o) Typical intracellular double membrane-bound biotrophic hyphae during late biotrophic phase, solid arrow indicates biotrophic hypha and dashed arrow plant derived membranes (32–34 hpi for Nirmala, 20–22 hpi in VRI-1). (n,p) GFP expressing hyphae within a few host cells during late biotrophic phase. (q,s) External view of lower stem and root of Nirmala and VRI-1 during biotrophic phase showing no visible symptoms. (r,t) Cross-section of roots of two varieties during biotrophic infection phase showing no necrotrophy. (u) Transition from biotrophy to necrotrophic phase (BNS) was characterized by development of thin, secondary necrotrophic hyphae from primary biotrophic hyphae. (v,w) External view of lower stem and root Nirmala nad VRI-1 still showing little or no symptoms. (x,y) Cross section of roots during BNS with trypan blue staining. BNS was delayed in the resistant variety being observed at 36–38 hpi in Nirmala and at 24–26 hpi in VRI-1. (Bar = 50 µm).
Necrotrophic phase of Macrophomina phaseolina infection in resistant sesame cultivar Nirmala (36–38 hpi onwards) and susceptible cultivar VRI-1 (24–26 hpi onwards). (a,c) Less extensive growth of GFP-expressing thin secondary hyphae in root tissues during necrotrophic phase in Nirmala (38 hpi) compared to VRI-1 (26 hpi). (b,d,e,h) Trypan blue staining and confocal microscopy using GFP-expressing mycelia show intercellular secondary necrotrophic hyphae (solid arrows) growing around the outline of the root cells and intracellular hyphae (dashed arrows) inside root cells in Nirmala and VRI-1. (f,i) Confocal microscopy showing scanty growth of GFP-expressing mycelia on root surface in Nirmala while in VRI-1 the root surface is covered by a mantle of fungal hyphae (72 hpi). (g,j) Less hyphal growth and microsclerotia (appearing as elongated black spots) on Nirmala roots compared to VRI-1 (96–120 hpi). (k,m) External symptoms of root in Nirmala (72 hpi) and VRI-1 (60 hpi) during early necrotrophic phase. (l,n) Trypan blue staining of root cross section showing less necrotrophic symptoms in Nirmala than VRI-1 at 96 hpi (black degenerated tissues shown by white arrow) (o) External symptoms on infected VRI-1 stem sowing typical charcoal rot symptom (120 hpi). (p) Confocal microscopy showing stem cortical cells heavily colonized with GFP-expressing secondary hyphae. Intercellular growth of hyphae is shown by solid arrow and intracellular growth by dashed arrow. (q,r) Longitudinal section of stem showing a group of xylem vessels colonized by GFP-expressing hyphae in necrotrophic phase of infection. (Bar = 50 µm). (s) Monitoring of disease progression by relative quantification of fungal biomass by q-PCR from DNA extracted from sesame roots infected with M. phaseolina. (t,u) Relative expression of M. phaseolina stage-specific marker genes for biotrophic stage (biotrophy associated secreted protein-BAS3) and necrotrophic stage (Necrosis inducing protein-NIP) in infected sesame roots at different time points post inoculation.
ROS metabolism and anti-oxidant enzyme activity in sesame during different phases of infection in resistant (Nirmala) and susceptible (VRI-1) varieties of sesame. (a) Quantitative estimation of H2O2 accumulation in different hours post inoculation (hpi). (b) Micrograph showing H2O2 accumulation in root cells after infection detected by DAB staining. (c) Quantitative estimation of lipid peroxidation (MDA equivalent). Quantitative estimation of enzyme activity of (d) superoxide dismutase (SOD) (e) ascorbate peroxidase (APX) (f) Catalase at different hpi. RT-qPCR analysis of the expression of antioxidant genes (g) SiRbOH (Sesamum indicum respiratory burst oxidase homolog), (h) SiSOD (S. indicum Superoxide dismutase) (i) SiGST = (S. indicum glutathione-s-transferase). (j) SiCysPI (S.indicum Cysteine protease inhibitor). Bars represent standard error (SE) of the mean (n = 3). Different letters indicate significant differences among treatments at p < 0.05, according to Duncan’s multiple range test.
Changes in secondary metabolites of sesame during different phases of infection by M. phaseolina. (a) Quantitative estimation of total phenol at different hpi. VRI-1 shows peak accumulation of phenolics at 12 hpi where as in Nirmala it the peak was at 36 hpi. (b) Micrographs showing less yellow autofluorescence for cell wall bound phenolics in VRI-1 cells than Nirmala at 36 hpi. (c) Quantitative estimation of PAL (phenylalanine ammonium lyase) activity at different hpi. (d) Quantitative estimation of flavonoids at different hpi. (e) Micrograph showing callose deposition in sesame cells at different hpi, with Nirmala showing earlier and more callose deposition than VRI-1. RT-qPCR analysis of the expression of genes for flavonoid biosynthesis: (f) SiIFR (S. indicum Isoflavone reductase) (g) SiFLM (S. indicum Flavonoid 3′-monooxygenase) (h) SiPCHY (S. indicum Paracoumaric-3-hydroxylase). Bars represent standard error (SE) of the mean (n = 3). Different letters indicate significant differences among treatments at p < 0.05, according to Duncan’s multiple range test.
RT-qPCR analysis of the expression of SA, calcium signaling genes and SA estimation in sesame during different phases of infection by M. phaseolina. (a) SiEDS1 (S. indicum Enhanced disease susceptibility 1) (b) SiNPR1 (Sesamum indicum Nonexpressor of PR genes1) (c) SiChi (S. indicum chitinase) (d) SiTLP (S. indicum Thaumatin like protein) (e) SiCaM (S. indicum calmodulin) (f) SiCDPK (Sesamum indicum calcium dependent protein kinase). Sesame eIF4A was used as an internal control. (g) GC-MS quantification of SA in the two varieties post inoculation with M.phaseolina. (h) GC-MS quantification of SA in the two varieties under mock inoculation. Bars represent standard error (SE) of the mean (n = 3). Different letters indicate significant differences among treatments at p < 0.05, according to Duncan’s multiple range test.
RT-qPCR analysis of the expression of JA/ET signaling genes and JA estimation in sesame during different phases of infection by M. phaseolina. (a) SiAOS (S. indicum Allene oxide synthase) (b) SiDef (Sesamum indicum Plant defensin) (c) SiSAM (S. indicum S-Adenosyl methionine synthetase) (d) SiERF (S. indicum Ethylene response factor) (e) SiAP2 (S. indicum EREBP/Apetalla2) (f) SiPDF1.2 (Sesamum indicum plant defensin 1.2) (g) SiJAZ (S. indicum Jasmonate ZIM-domain protein). Sesame eIF4A was used as an internal control. (h) GC-MS quantification of JA in the two varieties post inoculation with M. phaseolina. (i) GC-MS quantification of JA in the two varieties under mock inoculation. Bars represent standard error (SE) of the mean (n = 3). Different letters indicate significant differences among treatments at p < 0.05, according to Duncan’s multiple range test.
Phytohormone priming experiments by treatment of sesame (resistant variety Nirmala) with different phytohormones and their inhibitors to assay effect on tolerance against Macrophomina phaseolina. Plants treated with different chemicals 24 hrs prior to infection with M. phaseolina and pictures were taken 14 days post inoculation. (a) 100 µM Methyl jasmonate (b) 500 µM Ethepon (c) water (d) 200 µM salicylic acid (e) 100 µM DIECA (f) 20 µM Ibuprofen (IBU) (g) 20 mM STS (h) 100 µM Methyl jasmonate + 500 µM Ethepon (i) 20 mM STS + 100 µM DIECA (j) Disease index for inoculated sesame under different chemical treatment. Bars represent standard error (SE) of the mean (n = 3). Different letters indicate significant differences among treatments at p < 0.05, according to Duncan’s multiple range test. Confocal micrograph showing sesame roots infected with GFP-expressing M. phaseolina in (k) control (l) pre-treated with100 µM Methyl jasmonate + 500 µM Ethepon. (m) Pre-treated with 20 mM STS + 100 µM DIECA. RT-qPCR analysis of the expression of (n) SiAOS (o) SiSAM (p) SiAP2 under different treatments. (q) Schematic representation of proposed role of the sesame JA/ET genes in defense activation during M. phaseolina infection.
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