1/25/2019
Another widespread energy reserve form
β(2,6) Levan Fru Fru A Family of Novel Glycoside n β(2,1) Inulin Fru Fru Hydrolase Genes Originated n From Bacteria in Hessian Fly important storage carbohydrates in Genome over 45,000 plant species
Xiaoyan Cheng Degradation of 01/12/2019 Levan and Inulin ??
Hessian fly (Mayetiola destructor)
Levanase and inulinase found in plants and bacteria A significant pest of wheat
No levanase or inulinase has been found is animals or insects yet
(photo by Arlan Newby)
Sugar content in wheat
Non-feeding site
Feeding site
After infestation, sugar levels in wheat increase obviously
K5C: wheat without infestation, K5: wheat infested with Hessian fly K5Ci: non-feeding site sample of wheat without infestation, K5i: non-feeding site Glucose Storage sample of wheat infested with Hessian fly
1 1/25/2019
A Family of Levanases (MDLs) in Hessian Fly Genome I II III
Full length: 3 exons & 2 introns Glycosyl hydrolases family 32 domain MDLs with signal peptide except MDL6 and MDL10
Gene Expression of MDLs MDLs only share similarities with During Lifecycle of Hessian fly bacterial levanase and inulinase
1st-1 1st-3 2nd 3rd Pupae Male Female
Similarities of MDLs family Dissection of Hessian fly larva
MDL1 MDL2 MDL3 MDL4 MDL5 MDL6 MDL7 MDL8 MDL9 MDL10 MDL11 MDL12 MDL13 MDL14 MDL15 MDL2 56% - MDL3 64% 56% - MDL4 56% 96% 57% - MDL5 61% 56% 66% 56% - MDL6 67% 56% 70% 57% 67% - MDL7 92% 60% 57% 61% 59% 62% - MDL8 60% 99% 77% 99% 85% 78% 67% - MDL9 61% 51% 52% 51% 49% 53% 53% 69% - MDL10 63% 56% 64% 56% 65% 69% 58% 76% 54% - MDL11 66% 50% 57% 50% 55% 57% 55% 52% 48% 59% - MDL12 64% 56% 98% 57% 65% 70% 56% 79% 52% 63% 56% - MDL13 58% 96% 94% 94% 56% 57% 60% 98% 50% 57% 50% 56% - MDL14 95% 50% 44% 50% 47% 50% 69% 17% 49% 51% 55% 44% 50% - MDL15 83% 44% 47% 46% 44% 46% 46% none 51% 48% 44% 47% 44% 47% - MDL16 43% 46% 45% 46% 40% 48% 39% 61% 45% 48% 40% 45% 44% 35% 57%
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Gene Expression of MDLs Enzyme Kinetics of in Different Tissues of Hessian fly Recombinant Proteins Levanase Inulinase Sucrase
Vmax Vmax Vmax Km (mg/ml) Km (mg/ml) Km (mg/ml) (ug/min/ml) (ug/min/ml) (ug/min/ml) MDL2 0.24 6.54 0.11 3.05 0.83 12.46 MDL4 0.10 2.38 MDL5 0.53 8.48 0.22 4.11 2.27 19.29 MDL6 0.67 2.15 2.63 5.28 MDL7 0.96 3.37 0.46 1.98 10.23 34.80 MDL9 0.08 2.77 0.20 4.81 0.42 8.83 MDL11 1.03 6.35 3.49 9.13 9.35 28.72 MDL12 0.23 1.84 0.63 3.82 MDL13 1.00 3.63 1.96 7.30 MDL15 0.18 3.85 0.27 5.06
Some protein members have three kinds enzyme activities, some have two, and MDL4 has only inulinase activity
Enzyme activity of Hessian fly MDLs
Upper : enzymatic degradation activity assay for different sugar substrates Lower: TLC assay (thin layer chromatography) of reducing sugars released from degradation of substrates • Crude extract of Hessian fly shows three kinds of enzyme activities, such as levanase, inulinase and sucrase. • Enzyme activities show the highest levels in the larva than any other stages such as pupa, female adult and male adult. Especially, the 2nd larvae have the most enzyme activities than any other stages of Hessian fly. Different substrates: different reaction time, most favorable pH value and optimal temperature (MDL2 for example)
Recombinant MDLs proteins MDL5 in Hessian fly Larvae
MDL5 Control
Salivary Gland
Strong signal in both salivary gland and midgut Recombinant MDL proteins are soluble in E.coli cells (MDL2 for example) Midgut
DAPI MDL
Light Merged
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Fat Body
Weak or no signal in fat body and malpighian tubule
Malpighian Tubule
DAPI MDL
MDL5 Control Light Merged
Conclusions
1. MDLs family has 16 genes, sharing the glycosyl hydrolyses family 32 domain. 2. The levanase family of Hessian fly is horizontally transferred from Bacteria. 3. qPCR confirms the diversified gene expression patterns of MDLs family. 4. MDLs proteins show capability to digest big molecule sugars in Hessian fly, which contributes to Hessian fly making use of host wheat. 5. A clue is indicated that fructose functions importantly as an energy molecule in Hessian fly compared with traditional glucose.
Thank you
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