Identification of defense proteins in pearl millet seeds effective against Magnaporthe grisea
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Pearl millet leaf blast is caused by Magnaporthe grisea (Anamorph, Pyricularia grisea) has been recently emerged as devastating disease with economic significance in India. It is well-known that host plant resistance is the most economical strategy to effectively manage this disease; hence, identification of resistance sources for blast disease is important to incorporate resistance genes into elite breeding lines. On the other hand, fungal cell wall is a multi-layered, in which chitin and glucan are the major polysaccharide constituents (Figure 1). In this view, chitinases and glucanases gain significant attention as antifungal enzymes. These were produced as pathogenesis related (PR) hydrolses in plants with constitutive expression in seeds, leaves, flowers, tubers and induced upon pathogen invasion. They exert their defensive role by decomposing the fungal cell wall polysaccharides chitin and glucan into respective monomers as N-Acetyl D-glucosamine and D-glucose residues (Prasannath, 2017). Whereas, protease inhibitors (PIs) are known to participate in defensive role by inhibiting the extracellular protease activity secretes from actively growing fungal mycelia as well as cysteine proteases involved in the chitin synthase activity (Joshi et al., 1998). Hence, the present study is focused on the screening of chitinases, glucanases and cysteine protease inhibitors in ten pearl millet seed proteins with differential disease resistance and evaluation of their anti fungal efficacy against growth of P. grisea (Pg 45), prevalent isolate in Hyderabad, Telangana region.
![About ICRISAT: www.icrisat.org
ICRISAT’s scientific information: http://EXPLOREit.icrisat.org
Nov 2019
Identification of defense proteins in pearl millet
seeds effective against Magnaporthe grisea
Swathi Marri, Mahalingam Govindaraj, Rajan Sharma
International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru-502 324, Telangana, India
Rationale and Objective
Pearl millet leaf blast is caused by Magnaporthe grisea (Anamorph, Pyricularia grisea) has been
recently emerged as devastating disease with economic significance in India. It is well-known
that host plant resistance is the most economical strategy to effectively manage this disease;
hence, identification of resistance sources for blast disease is important to incorporate resistance
genes into elite breeding lines. On the other hand, fungal cell wall is a multi-layered, in which
chitin and glucan are the major polysaccharide constituents (Figure 1). In this view, chitinases and
glucanases gain significant attention as antifungal enzymes. These were produced as pathogenesis
related (PR) hydrolses in plants with constitutive expression in seeds, leaves, flowers, tubers and
induced upon pathogen invasion. They exert their defensive role by decomposing the fungal cell
wall polysaccharides chitin and glucan into respective monomers as N-Acetyl D-glucosamine and
D-glucose residues (Prasannath, 2017). Whereas, protease inhibitors (PIs) are known to participate in
defensive role by inhibiting the extracellular protease activity secretes from actively growing fungal
mycelia as well as cysteine proteases involved in the chitin synthase activity (Joshi et al., 1998).
Hence, the present study is focused on the screening of chitinases, glucanases and cysteine protease
inhibitors in ten pearl millet seed proteins with differential disease resistance and evaluation of their
anti fungal efficacy against growth of P. grisea (Pg 45), prevalent isolate in Hyderabad, Telangana region.
β-1,3 glucanases (r, 0.53) and cystatins (r, 0.65) are in positive significant correlation with the
disease resistance score (Pg 45) under glass house conditions (Table 1).
• Anti-fungal screenings of seed proteins (360 µg/ml) on oatmeal agar plates resulted in 22-40%
reduction in radial growth of Pg 45 as compared to control, which is represented in growth
curves (Fig. 2). The effective concentration for the 50% fungal growth inhibition (EC50
) was
identified as 400 and 600 μg/ml for resistant lines IP 21187 and ICMR 06444, respectively.
• The antifungal potency of tested seed proteins is further demonstrated by the 20-77% reduction
in dry weight of fungal biomass (Fig. 3A). A significant reduction in biomass dry weight was
observed with lines ICMR 06444 (77%) and IP 21187 (44%) as shown in Fig. 3B.
• Microspectrophotometric assays (A595
nm) using diluted spore suspension of Pg 45 in presence
of respective seed protein (16 μg) resulted in 24-83 % retardation in initial growth (Fig. 3C)
and spore germination of fungi (Fig. 3D).
• The identified PR hydrolases in the seed proteins chtinases and β-1,3 glucanases possibly
playing synergistic role in plant defense by decomposing the major structural polysaccharides
chitin [β-(1,4)-linked polymer of N-acetyl D-glucosamine, GlcNAc] and β-1,3 glucan [β-(1,3)-
linked polymer of D-glucose] of fungal cell wall (Prasannath, 2017). Whereas cysteine protease
inhibitors may play defense role by inhibiting the proteases involved in the chitin synthase
activity (Joshi et al., 1998).
PR hydrolases, cysteine PI activity in seed proteins and correlation with glasshouse screenings against
blast disease
Entry Name
Chitinase activity
(units/ml)
β-1,3 glucanase
actiivty (units/mg)
Cysteine PI activity
(units/mg)
Glasshouse screening
(0-9 scale*)
ICMB 9333 14.0 ± 0.8b
23.1 ± 0.7b
87.5 ± 2.5b
R
ICMB 95444 11.7 ± 0.9c
15.7 ± 0.4c
63.8 ± 3.6c
S
ICMB 97222 16.0 ± 0.6a
22.9 ± 0.6b
88.3 ± 2.5b
R
ICMB 01333 13.2 ± 1.2b
20.7 ± 0.7b
57.4 ± 1.2c
S
ICMB 02444 12.8 ± 0.2b
21.1 ± 1.1b
69.3 ± 0.8b
S
ICMR 06444 18.5 ± 0.4a
38.3 ± 2.5a
122.4 ± 6.4a
R
863BP2 13.2 ± 0.8b
22.5 ± 1.7b
66.7 ± 3.6c
S
ICMR 06222 15.6 ± 0.9a
23.1 ± 1.1b
71.3 ± 4.1b
R
ICMR 11003 15.8 ± 0.8a
23.8 ± 1.7b
71.7 ± 1.2b
R
IP 21187 18.2 ± 0.9a
47.8 ± 3.0a
123.8 ± 8.7a
R
Correlation (r) 0.81** 0.53** 0.65** ---
Different letters within column indicate significant variation among the millet lines. ** significant at 1% probability level
* 0-3 Resistant; 4-9 Susceptible
Figure 1 . Representative picture of foliar blast disease incidence on pearl millet plant. Inset: Structural
components of fungal cell wall.
Materials and Methods
Material selection: Ten pearl millet inbred (six seed
parents, three restorers and one germplasm derived)
having differential resistance response to foliar blast
disease were selected (863BP2, ICMB 9333, ICMB 95444,
ICMB 97222, ICMB 01333, ICMB 02444, ICMR 06444, ICMR
06222, ICMR 11003, IP 21187).
Isolate selection: The single spore culture of P. grisea
pathotype-isolate collected from pearl millet field located in
Patancheru (Pg 45), Hyderabad, India (Thakur et al., 2009).
Lab experiments: The protocols used in the present study are briefly explained in the flow chart.
Data Analysis: All the experiments were carried out three times each with three replications, and the
mean ± SE was reported by using Sigma plot 12.0 (Systat Software Inc., San Jose, CA). The Pearson’s
correlation coefficient among the traits was calculated using Sigma stat software.
Results
• The seed protein extrudes of inbreds exhibited significant pathogenesis related (PR-2 and PR-3)
hydrolase activity including β-1,3 glucanases (16-47.8 Units/mg protein) and chitinases (12-18.2
Units/ml) as well as cysteine protease inhibitor (PR-6) activity against papain (57-123 Units/mg
protein) as shown in Table 1. The activity levels of PR hydrolases chitinases (r, 0.81),
Conclusion
Pearl millet seed has adequate levels of PR hydrolases such as chitinases, β-1,3 glucanases and
cysteine protease inhibitors and can be explored to capture variability in breeding populations
and germplasm. The recognized PR proteins will be helpful as biochemical markers to screen the
differential resistance against foliar blast in pearl millet and also may useful for the exploitation of
novel defense strategies helpful in resistance breeding.
Acknowledgements
This research study was supported by N-PDF funding from the Science and Engineering Research
Board (SERB), a statutory body of Department of Science and Technology (DST), Government of India
is greatly acknowledged.
References
Broekaert WF. et al., (1990). FEMS Microbiol Lett, 69: 55-60.
Cole MD. (1994). Biochem syst & Eco, 22: 837-856.
Ferrari AR. et al., (2014). Biotechnol Biofuels, 7: 37.
Filippova Y. et al., (1984). Anal Biochem, 143: 293-297.
Joshi BN. et al., (1998). Biochem Biophys Res Comm, 246: 382–387.
Koga D. (1988). Agric Biol Chem, 52: 2091–93.
Prasannath K. (2017). J Agric Sci 11: 38.
Thakur, RP. (2009). J SAT Agric Res, 7: 1-5.
Figure 2. Antifungal efficacy of seed proteins (360 μg/ml) A. 1-5 B. 6-10 on radial growth of P. grisea.
Statistical difference among growth curves at P<0.05 are shown with different symbols.
Crude protein extraction
Fungal zone inhibition
assays (Cole, 1994)
Microspectrophotometric
assays (Broekaert et al., 1990)
Chitinase assay
(Ferrai et al., 2014)
Cysteine protease
inhibition assay
(Fillipova et al., 1984)
Pearl millet seeds
Antifungal screening
*(% control)
Biochemical
characterization
*control - fungal culture without addition of test sample
β-1,3 Glucanase assay
(Koga et al., 1988)
Methodology followed for the study.
Methodology followed for the study.
B
Figure 3. Inhibitory effect of seed proteins against growth of P. grisea evident by A. dry weight reduction of
fungal biomass at 0.001% w/v, B. restoration of culture nutritional media with gradual increase of ICMR
06444. C. Microspectrophotometric assays (A595) in presence of seed proteins (16 μg). D. Retardation
of spore germination (ICMR 06444, 16 μg) One way ANOVA test was performed (Tukey method) and the
statistical difference at P<0.05 are shown with ‘#’ symbol
A
Spores (control)
Spores + seed protein (18 h)
0 h 18 h
1 2 3
1 Control (PDB + Fungal disc)
2.Seed protein (400 µg)
3. Seed protein (800 µg)
B
D
Email: mswathi.win@gmail.com](https://image.slidesharecdn.com/jobno-200619074510/85/Identification-of-defense-proteins-in-pearl-millet-seeds-effective-against-Magnaporthe-grisea-1-320.jpg)
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Identification of defense proteins in pearl millet seeds effective against Magnaporthe grisea
- 1. About ICRISAT: www.icrisat.org ICRISAT’s scientific information: http://EXPLOREit.icrisat.org Nov 2019 Identification of defense proteins in pearl millet seeds effective against Magnaporthe grisea Swathi Marri, Mahalingam Govindaraj, Rajan Sharma International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru-502 324, Telangana, India Rationale and Objective Pearl millet leaf blast is caused by Magnaporthe grisea (Anamorph, Pyricularia grisea) has been recently emerged as devastating disease with economic significance in India. It is well-known that host plant resistance is the most economical strategy to effectively manage this disease; hence, identification of resistance sources for blast disease is important to incorporate resistance genes into elite breeding lines. On the other hand, fungal cell wall is a multi-layered, in which chitin and glucan are the major polysaccharide constituents (Figure 1). In this view, chitinases and glucanases gain significant attention as antifungal enzymes. These were produced as pathogenesis related (PR) hydrolses in plants with constitutive expression in seeds, leaves, flowers, tubers and induced upon pathogen invasion. They exert their defensive role by decomposing the fungal cell wall polysaccharides chitin and glucan into respective monomers as N-Acetyl D-glucosamine and D-glucose residues (Prasannath, 2017). Whereas, protease inhibitors (PIs) are known to participate in defensive role by inhibiting the extracellular protease activity secretes from actively growing fungal mycelia as well as cysteine proteases involved in the chitin synthase activity (Joshi et al., 1998). Hence, the present study is focused on the screening of chitinases, glucanases and cysteine protease inhibitors in ten pearl millet seed proteins with differential disease resistance and evaluation of their anti fungal efficacy against growth of P. grisea (Pg 45), prevalent isolate in Hyderabad, Telangana region. β-1,3 glucanases (r, 0.53) and cystatins (r, 0.65) are in positive significant correlation with the disease resistance score (Pg 45) under glass house conditions (Table 1). • Anti-fungal screenings of seed proteins (360 µg/ml) on oatmeal agar plates resulted in 22-40% reduction in radial growth of Pg 45 as compared to control, which is represented in growth curves (Fig. 2). The effective concentration for the 50% fungal growth inhibition (EC50 ) was identified as 400 and 600 μg/ml for resistant lines IP 21187 and ICMR 06444, respectively. • The antifungal potency of tested seed proteins is further demonstrated by the 20-77% reduction in dry weight of fungal biomass (Fig. 3A). A significant reduction in biomass dry weight was observed with lines ICMR 06444 (77%) and IP 21187 (44%) as shown in Fig. 3B. • Microspectrophotometric assays (A595 nm) using diluted spore suspension of Pg 45 in presence of respective seed protein (16 μg) resulted in 24-83 % retardation in initial growth (Fig. 3C) and spore germination of fungi (Fig. 3D). • The identified PR hydrolases in the seed proteins chtinases and β-1,3 glucanases possibly playing synergistic role in plant defense by decomposing the major structural polysaccharides chitin [β-(1,4)-linked polymer of N-acetyl D-glucosamine, GlcNAc] and β-1,3 glucan [β-(1,3)- linked polymer of D-glucose] of fungal cell wall (Prasannath, 2017). Whereas cysteine protease inhibitors may play defense role by inhibiting the proteases involved in the chitin synthase activity (Joshi et al., 1998). PR hydrolases, cysteine PI activity in seed proteins and correlation with glasshouse screenings against blast disease Entry Name Chitinase activity (units/ml) β-1,3 glucanase actiivty (units/mg) Cysteine PI activity (units/mg) Glasshouse screening (0-9 scale*) ICMB 9333 14.0 ± 0.8b 23.1 ± 0.7b 87.5 ± 2.5b R ICMB 95444 11.7 ± 0.9c 15.7 ± 0.4c 63.8 ± 3.6c S ICMB 97222 16.0 ± 0.6a 22.9 ± 0.6b 88.3 ± 2.5b R ICMB 01333 13.2 ± 1.2b 20.7 ± 0.7b 57.4 ± 1.2c S ICMB 02444 12.8 ± 0.2b 21.1 ± 1.1b 69.3 ± 0.8b S ICMR 06444 18.5 ± 0.4a 38.3 ± 2.5a 122.4 ± 6.4a R 863BP2 13.2 ± 0.8b 22.5 ± 1.7b 66.7 ± 3.6c S ICMR 06222 15.6 ± 0.9a 23.1 ± 1.1b 71.3 ± 4.1b R ICMR 11003 15.8 ± 0.8a 23.8 ± 1.7b 71.7 ± 1.2b R IP 21187 18.2 ± 0.9a 47.8 ± 3.0a 123.8 ± 8.7a R Correlation (r) 0.81** 0.53** 0.65** --- Different letters within column indicate significant variation among the millet lines. ** significant at 1% probability level * 0-3 Resistant; 4-9 Susceptible Figure 1 . Representative picture of foliar blast disease incidence on pearl millet plant. Inset: Structural components of fungal cell wall. Materials and Methods Material selection: Ten pearl millet inbred (six seed parents, three restorers and one germplasm derived) having differential resistance response to foliar blast disease were selected (863BP2, ICMB 9333, ICMB 95444, ICMB 97222, ICMB 01333, ICMB 02444, ICMR 06444, ICMR 06222, ICMR 11003, IP 21187). Isolate selection: The single spore culture of P. grisea pathotype-isolate collected from pearl millet field located in Patancheru (Pg 45), Hyderabad, India (Thakur et al., 2009). Lab experiments: The protocols used in the present study are briefly explained in the flow chart. Data Analysis: All the experiments were carried out three times each with three replications, and the mean ± SE was reported by using Sigma plot 12.0 (Systat Software Inc., San Jose, CA). The Pearson’s correlation coefficient among the traits was calculated using Sigma stat software. Results • The seed protein extrudes of inbreds exhibited significant pathogenesis related (PR-2 and PR-3) hydrolase activity including β-1,3 glucanases (16-47.8 Units/mg protein) and chitinases (12-18.2 Units/ml) as well as cysteine protease inhibitor (PR-6) activity against papain (57-123 Units/mg protein) as shown in Table 1. The activity levels of PR hydrolases chitinases (r, 0.81), Conclusion Pearl millet seed has adequate levels of PR hydrolases such as chitinases, β-1,3 glucanases and cysteine protease inhibitors and can be explored to capture variability in breeding populations and germplasm. The recognized PR proteins will be helpful as biochemical markers to screen the differential resistance against foliar blast in pearl millet and also may useful for the exploitation of novel defense strategies helpful in resistance breeding. Acknowledgements This research study was supported by N-PDF funding from the Science and Engineering Research Board (SERB), a statutory body of Department of Science and Technology (DST), Government of India is greatly acknowledged. References Broekaert WF. et al., (1990). FEMS Microbiol Lett, 69: 55-60. Cole MD. (1994). Biochem syst & Eco, 22: 837-856. Ferrari AR. et al., (2014). Biotechnol Biofuels, 7: 37. Filippova Y. et al., (1984). Anal Biochem, 143: 293-297. Joshi BN. et al., (1998). Biochem Biophys Res Comm, 246: 382–387. Koga D. (1988). Agric Biol Chem, 52: 2091–93. Prasannath K. (2017). J Agric Sci 11: 38. Thakur, RP. (2009). J SAT Agric Res, 7: 1-5. Figure 2. Antifungal efficacy of seed proteins (360 μg/ml) A. 1-5 B. 6-10 on radial growth of P. grisea. Statistical difference among growth curves at P<0.05 are shown with different symbols. Crude protein extraction Fungal zone inhibition assays (Cole, 1994) Microspectrophotometric assays (Broekaert et al., 1990) Chitinase assay (Ferrai et al., 2014) Cysteine protease inhibition assay (Fillipova et al., 1984) Pearl millet seeds Antifungal screening *(% control) Biochemical characterization *control - fungal culture without addition of test sample β-1,3 Glucanase assay (Koga et al., 1988) Methodology followed for the study. Methodology followed for the study. B Figure 3. Inhibitory effect of seed proteins against growth of P. grisea evident by A. dry weight reduction of fungal biomass at 0.001% w/v, B. restoration of culture nutritional media with gradual increase of ICMR 06444. C. Microspectrophotometric assays (A595) in presence of seed proteins (16 μg). D. Retardation of spore germination (ICMR 06444, 16 μg) One way ANOVA test was performed (Tukey method) and the statistical difference at P<0.05 are shown with ‘#’ symbol A Spores (control) Spores + seed protein (18 h) 0 h 18 h 1 2 3 1 Control (PDB + Fungal disc) 2.Seed protein (400 µg) 3. Seed protein (800 µg) B D Email: mswathi.win@gmail.com