Example 7
As shown in some of the earlier examples, endophytic microbes described herein are capable of conferring significant beneficial traits on the inoculated agricultural plant. In order to explore the pathways augmented or otherwise modified by the endophyte, we performed proteomic analysis on extracts of wheat, maize and soy plants grown on water agar. Sterilized wheat, maize and soy seeds were either mock-inoculated with R2A medium (formulation control), or inoculated with selected endophytes using conditions previously described. The seeds were subjected to the growth parameters as summarized below (Table 15).
TABLE 15
Samples used for proteomic measurements.
Sample #CropTestCondition
1Wheat (Briggs)Formulation controlNormal
2Wheat (Briggs)SYM00218Normal
3Wheat (Briggs)Formulation controlHeat
4Wheat (Briggs)SYM00011Heat
5Wheat (Briggs)SYM00016Heat
6Wheat (Briggs)SYM00057BHeat
7Maize (40R73)Formulation controlNormal
8Maize (40R73)SYM00057BNormal
9Soy (Butterbean)Formulation controlNormal
10Soy (Butterbean)SYM00057BNormal
11Soy (Butterbean)SYM00498Normal
12Soy (Butterbean)SYM00596Normal
13Soy (Butterbean)Formulation controlNormal
14Soy (Butterbean)SYM00002Normal
15Soy (Butterbean)SYM00046Normal
16Soy (Butterbean)SYM00052Normal
17Soy (Butterbean)Formulation controlNormal
18Soy (Butterbean)SYM00218Normal
19Soy (Butterbean)SYM00508Normal
20Soy (Butterbean)SYM00940Normal
Sample Collection
Maize and wheat: After 4 days of growth, 12 whole seedlings (including roots, seeds and hypocotyls) per treatment were collected in a 50 mL falcon tube using sterile forceps and immediately snap-frozen in liquid nitrogen to minimize protein degradation and proteomic changes during sample collection (such as wound responses from using the forceps). The frozen samples were then homogenized using a pestle and mortar previously cooled in liquid nitrogen and transferred to a 15 mL falcon tube on dry ice. The homogenized samples were stored at −80° C. until further processing.
Soy: After 5 days of growth, the roots of 27 seedlings per treatment were collected in a 50 mL falcon tube using flamed forceps and immediately snap-frozen in liquid nitrogen to minimize protein degradation and proteomic changes during sample collection (such as wound responses from using the forceps). The frozen samples were then homogenized using a pestle and mortar previously cooled in liquid nitrogen and transferred to a 15 mL falcon tube on dry ice. The homogenized samples were stored at −80° C. until further processing.
Sample Preparation
1 mL of 5% SDS 1 mM DTT was added to 1 mL of homogenized tissue and the samples were boiled for 5 m. The samples were cooled on ice and 2 mL of 8M urea solution was added. The samples were spun for 20 m at 14,000 rpm and the soluble phase recovered. A 25% volume of 100% TCA solution was added to the soluble phase, left on ice for 20 m and centrifuged for 10 m at 14,000 rpm. The protein pellet was washed twice with ice-cold acetone and solubilized in 125 μL 0.2M NaOH and neutralized with 125 μL of 1M Tris-Cl pH 8.0. Protein solutions were diluted in THE (50 mM Tris-Cl pH8.0, 100 mM NaCl, 1 mM EDTA) buffer. RapiGest SF reagent (Waters Corp., Milford, Mass.) was added to the mix to a final concentration of 0.1% and samples were boiled for 5 min. TCEP (Tris (2-carboxyethyl) phosphine) was added to 1 mM (final concentration) and the samples were incubated at 37° C. for 30 min. Subsequently, the samples were carboxymethylated with 0.5 mg ml−1 of iodoacetamide for 30 min at 37° C. followed by neutralization with 2 mM TCEP (final concentration). Proteins samples prepared as above were digested with trypsin (trypsin:protein ratio—1:50) overnight at 37° C. RapiGest was degraded and removed by treating the samples with 250 mM HCl at 37° C. for 1 h followed by centrifugation at 14,000 rpm for 30 min at 4° C. The soluble fraction was then added to a new tube and the peptides were extracted and desalted using Aspire RP30 desalting columns (Thermo Scientific). The trypsinized samples were labeled with isobaric tags (iTRAQ, ABSCIEX, Ross et al 2004), where each sample was labeled with a specific tag to its peptides.
Mass Spectrometry Analysis
Each set of experiments (samples 1-6; 7, 8; 9-12; 13-16; 17-20) was then pooled and fractionated using high pH reverse phase chromatography (HPRP-Xterra C18 reverse phase, 4.6 mm×10 mm 5 μm particle (Waters)). The chromatography conditions were as follows: the column was heated to 37° C. and a linear gradient from 5-35% B (Buffer A-20 mM ammonium formate pH10 aqueous, Buffer B-20 mM ammonium formate pH10 in 80% ACN-water) was applied for 80 min at 0.5 ml min−1 flow rate. A total of 30 fractions of 0.5 ml volume where collected for LC-MS/MS analysis. Each of these fractions was analyzed by high-pressure liquid chromatography (HPLC) coupled with tandem mass spectroscopy (LC-MS/MS) using nano-spray ionization. The nanospray ionization experiments were performed using a TripleTof 5600 hybrid mass spectrometer (AB SCIEX Concord, Ontario, Canada)) interfaced with nano-scale reversed-phase HPLC (Tempo, Applied Biosystems (Life Technologies), CA, USA) using a 10 cm-180 micron ID glass capillary packed with 5 μm C18 Zorbax™ beads (Agilent Technologies, Santa Clara, Calif.). Peptides were eluted from the C18 column into the mass spectrometer using a linear gradient (5-30%) of ACN (Acetonitrile) at a flow rate of 550 μl min−1 for 100 min. The buffers used to create the ACN gradient were: Buffer A (98% H2O, 2% ACN, 0.2% formic acid, and 0.005% TFA) and Buffer B (100% ACN, 0.2% formic acid, and 0.005% TFA). MS/MS data were acquired in a data-dependent manner in which the MS1 data was acquired for 250 ms at m/z of 400 to 1250 Da and the MS/MS data was acquired from m/z of 50 to 2,000 Da. For Independent data acquisition (IDA) parameters MS1-TOF 250 ms, followed by 50 MS2 events of 25 ms each. The IDA criteria, over 200 counts threshold, charge state +2-4 with 4 s exclusion. Finally, the collected data were analyzed using Protein Pilot 4.0 (AB SCIEX) for peptide identifications and quantification.
Results for Plant Inoculation Tests
The proteomics analysis of wheat inoculated with endophytic bacteria (SYM00011, SYM00016B and SYM00057B) grown under heat stress, maize inoculated with SYM00057B grown under normal condition and soy inoculated with (SYM00057B, SYM00596, SYM00052, SYM00002, SYM00046, SYM00218, SYM00508 and SYM00940) revealed three major pathways augmented or otherwise modified by the endophyte: growth promotion, resistance against oxidative stress and mechanisms involved in symbiosis enhancement (Tables 16, 17 and 18). In some embodiments, synthetic endophyte-plant combinations exhibit alteration of multiple plant protein abundance, particularly proteins involved in stress resistance. In some embodiments, alterations comprise upregulation relative to reference agricultural plants of the following polypeptides: gi|351723089/Chalcone-flavonone isomerase 1A and gi|351723125/Glutathione S-transferase GST 24 or gi|351723089/Chalcone-flavonone isomerase 1A and gi|358248196/Polygalacturonase inhibitor 1-like precursor or gi|351723089/Chalcone-flavonone isomerase 1A and gi|359807261/Soyasaponin III rhamnosyltransferase or gi|351723089/Chalcone-flavonone isomerase 1A and gi|571453722/Programmed cell death protein 4 or gi|356536151/Alpha-L-fucosidase 2-like and gi|351723089/Chalcone-flavonone isomerase 1A or gi|356536151/Alpha-L-fucosidase 2-like and gi|351723125/Glutathione S-transferase GST 24 or gi|356536151/Alpha-L-fucosidase 2-like and gi|356559376/26S protease regulatory subunit 7-like or gi|356536151/Alpha-L-fucosidase 2-like and gi|358248196/Polygalacturonase inhibitor 1-like precursor or gi|356536151/Alpha-L-fucosidase 2-like and gi|358249064/Uncharacterized protein LOC100795412 or gi|356536151/Alpha-L-fucosidase 2-like and gi|359807261/Soyasaponin III rhamnosyltransferase or gi|356536151/Alpha-L-fucosidase 2-like and gi|571453722/Programmed cell death protein 4 or gi|356559376/26S protease regulatory subunit 7-like and gi|351723089/Chalcone-flavonone isomerase 1A or gi|356559376/26S protease regulatory subunit 7-like and gi|351723125/Glutathione S-transferase GST 24 or gi|356559376/26S protease regulatory subunit 7-like and gi|358248196/Polygalacturonase inhibitor 1-like precursor or gi|356559376/26S protease regulatory subunit 7-like and gi|359807261/Soyasaponin III rhamnosyltransferase or gi|356559376/26S protease regulatory subunit 7-like and gi|571453722/Programmed cell death protein 4 or gi|358248196/Polygalacturonase inhibitor 1-like precursor and gi|351723125/Glutathione S-transferase GST 24 or gi|358249064/Uncharacterized protein LOC100795412 and gi|351723089/Chalcone-flavonone isomerase 1A or gi|358249064/Uncharacterized protein LOC100795412 and gi|351723125/Glutathione 5-transferase GST 24 or gi|358249064/Uncharacterized protein LOC100795412 and gi|356559376/26S protease regulatory subunit 7-like or gi|358249064/Uncharacterized protein LOC100795412 and gi|358248196/Polygalacturonase inhibitor 1-like precursor or gi|358249064/Uncharacterized protein LOC100795412 and gi|359807261/Soyasaponin III rhamnosyltransferase or gi|358249064/Uncharacterized protein LOC100795412 and gi|571453722/Programmed cell death protein 4 or gi|359807261/Soyasaponin III rhamnosyltransferase and gi|351723125/Glutathione S-transferase GST 24 or gi|359807261/Soyasaponin III rhamnosyltransferase and gi|358248196/Polygalacturonase inhibitor 1-like precursor or gi|571453722/Programmed cell death protein 4 and gi|351723125/Glutathione S-transferase GST 24 or gi|571453722/Programmed cell death protein 4 and gi|358248196/Polygalacturonase inhibitor 1-like precursor or gi|571453722/Programmed cell death protein 4 and gi|359807261/Soyasaponin III rhamnosyltransferase.
In some embodiments, synthetic endophyte-plant combinations exhibit altered abundance of two or more plant proteins that are each involved in distinct beneficial activities in the plant (e.g., plant growth promotion, resistance to stress, or symbiosis). In some embodiments, alterations comprise upregulation relative to reference agricultural plants of the following polypeptides: gi|351723615/Aspartate aminotransferase glyoxysomal isozyme AAT1 precursor and gi|356508869/Lysosomal alpha-mannosidase-like or gi|351723615/Aspartate aminotransferase glyoxysomal isozyme AAT1 precursor and gi|571436840/Pectinesterase/pectinesterase inhibitor 18-like or gi|356536151/Alpha-L-fucosidase 2-like and gi|351723615/Aspartate aminotransferase glyoxysomal isozyme AAT1 precursor or gi|356536151/Alpha-L-fucosidase 2-like and gi|356508869/Lysosomal alpha-mannosidase-like or gi|356536151/Alpha-L-fucosidase 2-like and gi|358248512/Uncharacterized protein LOC100796978 or gi|356536151/Alpha-L-fucosidase 2-like and gi|571436840/Pectinesterase/pectinesterase inhibitor 18-like or gi|358248512/Uncharacterized protein LOC100796978 and gi|356508869/Lysosomal alpha-mannosidase-like or gi|358248512/Uncharacterized protein LOC100796978 and gi|571436840/Pectinesterase/pectinesterase inhibitor 18-like or gi|358249064/Uncharacterized protein LOC100795412 and gi|351723615/Aspartate aminotransferase glyoxysomal isozyme AAT1 precursor or gi|358249064/Uncharacterized protein LOC100795412 and gi|356508869/Lysosomal alpha-mannosidase-like or gi|358249064/Uncharacterized protein LOC100795412 and gi|358248512/Uncharacterized protein LOC100796978 or gi|358249064/Uncharacterized protein LOC100795412 and gi|571436840/Pectinesterase/pectinesterase inhibitor 18-like.
In some embodiments, synthetic endophyte-plant combinations exhibit altered abundance of two or more plant proteins that are each involved in distinct beneficial activities in the plant (e.g., plant growth promotion, resistance to stress, or symbiosis). In some embodiments, alterations comprise upregulation relative to reference agricultural plants of the following polypeptides: gi|351723615/Aspartate aminotransferase glyoxysomal isozyme AAT1 precursor, gi|356505888/RuBisCO-associated protein-like, gi|571470673/Peroxisomal (S)-2-hydroxy-acid oxidase GLO1, gi|356563759/L-arabinokinase-like isoform 1, gi|356558075/Cytosolic triosephosphate isomerase, gi|351724869/Uncharacterized protein LOC100306662, gi|356562473/Xylose isomerase [Glycine max], gi|356563694/ATP synthase protein MI25-like, gi|358248540/Uncharacterized protein LOC100778245, gi|359807469/Molecule involved in rac1 cell signaling, gi|358249004/Uncharacterized protein LOC100792337, gi|571466979/Polyphenol oxidase A1, chloroplastic, gi|356534524/40S ribosomal protein S17-like in combination with down-regulation relative to reference agricultural plants of the following polypeptides: gi|571440773/Villin-3-like isoform X4, gi|356509275/Glutamate synthase [NADH], amyloplastic-like, gi|356506969/Coiled-coil domain-containing protein 124-like, gi|571521870/Nuclear pore anchor-like, gi|571440773/Villin-3-like isoform X4, gi|356509275/Glutamate synthase [NADH], amyloplastic-like, gi|356506969/Coiled-coil domain-containing protein 124-like, gi|571521870/Nuclear pore anchor-like.
In some embodiments, synthetic endophyte-plant combinations exhibit altered abundance of two or more plant proteins that are each involved in distinct beneficial activities in the plant (e.g., plant growth promotion, resistance to stress, or symbiosis). In some embodiments, alterations comprise down regulation relative to reference agricultural plants of the following polypeptides: gi|356506190/Transketolase, chloroplastic and gi|356509275/Glutamate synthase [NADH], amyloplastic-like or gi|356506190/Transketolase, chloroplastic and gi|356516458/Staphylococcal nuclease domain-containing or gi|356506190/Transketolase, chloroplastic and gi|356533407/Embryonic protein DC-8-like or gi|356506190/Transketolase, chloroplastic and gi|356535993/Beta-conglycinin, alpha chain or gi|356506190/Transketolase, chloroplastic and gi|356575855/Beta-conglycinin, beta chain-like or gi|356506190/Transketolase, chloroplastic and gi|571477629/Low-temperature-induced 65 kDa prot. or gi|356509275/Glutamate synthase [NADH], amyloplastic-like and gi|356516458/Staphylococcal nuclease domain-containing or gi|356509275/Glutamate synthase [NADH], amyloplastic-like and gi|356533407/Embryonic protein DC-8-like or gi|356509275/Glutamate synthase [NADH], amyloplastic-like and gi|356535993/Beta-conglycinin, alpha chain or gi|356509275/Glutamate synthase [NADH], amyloplastic-like and gi|356575855/Beta-conglycinin, beta chain-like or gi|356509275/Glutamate synthase [NADH], amyloplastic-like and gi|571477629/Low-temperature-induced 65 kDa prot. or gi|356533407/Embryonic protein DC-8-like and gi|356535993/Beta-conglycinin, alpha chain or gi|356533407/Embryonic protein DC-8-like and gi|356575855/Beta-conglycinin, beta chain-like or gi|356533407/Embryonic protein DC-8-like and gi|571477629/Low-temperature-induced 65 kDa prot. or gi|356575855/Beta-conglycinin, beta chain-like and gi|356535993/Beta-conglycinin, alpha chain or gi|571477629/Low-temperature-induced 65 kDa prot. and gi|356535993/Beta-conglycinin, alpha chain or gi|571477629/Low-temperature-induced 65 kDa prot. and gi|356575855/Beta-conglycinin, beta chain-like.
TABLE 16
Proteins showing differential levels of expression under heat stress in endophyte-inoculated
wheat (cv. Briggs) seedlings relative to not-inoculated control seedlings.
ALTERED PROTEIN PRODUCTION IN RESPONSE TO INOCULATION WITH BACTERIAL SEED ENDOPHYTES
Ratio Treatment/Control
Growth promotion(fold changes)
Accession #Gene namePathwaySYM00011SYM00016BSYM00057B
gi|474293349Acid beta-fructofuranosidasemobilization of sucrose0.5-1 1-21-2
gi|473798701ATP synthase subunit beta, mitochondrialATP synthesis1-21-2
gi|473945263Fructan 1-exohydrolasemobilization of fructans1-2
gi|473798921Glutamine synthetase cytosolic isozyme 1-2Amino acid biosynthesis1-21-2
gi|474427549Dynamin-related protein 1ECell division1-21-21-2
gi|474154210Histone H1Cell division1-21-21-2
gi|474396419Histone H1Cell division1-21-2
gi|474315053Histone H2ACell division1-21-2>2
gi|474114390Histone H2ACell division1-2
gi|474408930Histone H2A.1Cell division1-2>2
gi|474247555Protein H2A.7Cell division1-20.5-1
gi|474400621Histone H4Cell division1-21-2
gi|474160133Serine carboxypeptidase-like proteinAmino acid release1-21-21-2
gi|474397165Serine carboxypeptidase-like 51Amino acid release>21-2
gi|474449933Pectinesterase 1Cell wall remodeling1-2>2
gi|474193958Peptidyl-prolyl cis-trans isomerase CYP40Juvenile phase of vegetative1-2>2>2
development
gi|473956589Ribonucleoside-diphosphate reductaseDNA synthesis0.1-0.50.1-0.5>10
gi|474326915Villin-4Cell elongation>2>10 >2
gi|474156626Glutenin, low molecular weight subunitProtein storage - affected by heat1-21-2
Ratio Treatment/Control
Resistance against abiotic stress(fold changes)
Accession #Gene nameFunctionSYM00011SYM00016BSYM00057B
gi|474449933Pectinesterase 1Resistance to drought1-2>2
gi|474381202Peroxiredoxin Q, chloroplasticResistance to oxidative stress0.5-1 0.5-1 >2
gi|474299547Glutathione S-transferase DHAR3,Resistance to oxidative stress1-21-2>2
chloroplastic
gi|474276683Peroxidase 12Resistance to oxidative stress1-21-21-2
gi|4744145793-hydroxybenzoate 6-hydroxylase 1Degradation of toxic organic compounds1-2>21-2
gi|474323467BAHD acyltransferase DCRCutin formation - dessication resistance1-21-20.1-0.5
gi|4739996265′-methylthioadenosine/S-Negative feedback on ethylene0.5-1 0.5-1 0.5-1
adenosylhomocysteine nucleosidaseproduction
gi|474326305Aldehyde dehydrogenase family 2 memberControls acetaldehyde accumulation0.5-1 0.5-1 0.5-1
C4
gi|474041937putative protein phosphatase 2C 45Regulates ABA signaling0.5-1
gi|473894812DEAD-box ATP-dependent RNA helicase 40mRNA decay and ribosome biogenesis0.1-0.5
Ratio Treatment/Control
Symbiosis enhancement(fold changes)
Accession #Gene nameFunctionSYM00011SYM00016BSYM00057B
gi|474407144Enolase 1Glycolisis of sugars required by0.5-10.5-1
endophyte
gi|474119301Protochlorophyllide reductase B,Affected by symbiosis0.5-1
chloroplastic
gi|474213532Elicitor-responsive protein 1Microbe response signaling0.5-10.5-11-2
TABLE 17
Proteins showing differential levels of expression under normal condition in endophyte-inoculated
maize (Blue River Hybrids 40R73) seedlings relative to non-inoculated control seedlings.
ALTERED PROTEIN PRODUCTION IN RESPONSE TO INOCULATION WITH BACTERIAL SEED ENDOPHYTES
Ratio Treatment/Control
Growth promotion(fold changes)
Accession #Gene namePathwaySYM00057B vs. control
gi|413950290putative peptidyl-prolyl cis-trans isomeraseOrgan development >2-fold
gi|414876902ATP-dependent Clp protease proteolytic subunitChloroplast component >2-fold
gi|413948820Translation elongation factor Tu isoform 3Protein biosynthesis 1-2 fold
gi|414878150Chaperone protein dnaJ 15Positive gravitropism<0.5-fold
gi|413954599translation elongation/initiation factorEmbryo development ends<0.5-fold
seed dormancy
Resistance against abiotic stress
SYM00057B vs. control
Accession #Gene nameFunction(fold change)
gi|414867473Glutathione S-transferase GSTU6Resistance to oxidative 1-2
stress
gi|414876903Calmodulin2ABA-induced antioxidant<0.5
defense
gi|413920116Ras protein Rab-18ABA inducible,0.5-1
accumulates in cold stress
gi|413926351DNA repair protein RAD23-1 isoform 3Nucleotide-excision repair0.5-1
Symbiosis enhancement
SYM00057B vs.control
Accession #Gene nameFunction(fold change)
gi|413920282Hydroquinone glucosyltransferaseUpregulated in Rhizobia>10
symbiosis
gi|413939151replication factor C subunit 3Negative regulation of>10
defense response
gi|413946904NEDD8-activating enzyme E1 catalytic subunitProtein neddylation ->10
microbe response
gi|413951445delta3,5-delta2,4-dienoyl-CoA isomerasePeroxisome component ->10
defense
gi|413925737Proteasome subunit alpha typeResponse to compatible>2
symbiotic bacteria
gi|413957021Ras protein RHN1Legume homolog involved>2
in nodulation
gi|414875813Early nodulin 20Root nodule formation>2
gi|414886632Putative plant regulator RWP-RK family proteinNodule inception protein1-2
gi|413955359putative metacaspase family proteinProgrammed cell death0.5-1
regulation
gi|413920552win1Defense response to<0.5
bacteria and fungi
gi|413948744protein brittle-1Response to nematodes<0.5
gi|414869634Proteasome subunit beta typeRegulation of0.5-1
hypersensitive response
TABLE 18
Proteins showing differential levels of expression under normal condition in endophyte-inoculated soybean seedlings relative to non-inoculated control seedlings.
ALTERED PROTEIN PRODUCTION IN RESPONSE TO INOCULATION WITH BACTERIAL SEED ENDOPHYTES
Growth promotionRatio Treatment/Control (fold change)
Accession numberGene namePathwaySYM00057BSYM00596SYM00052SYM00002SYM00046SYM00218SYM00508SYM00940
gi|351726277ArginaseProline biosynthesis1-21-2
gi|359806184Uncharacterized proteinRNA binding domain>2>2
LOC100812934
gi|359806656Stem 31 kDa glycoproteinStorage>21-21-21-2
precursor
gi|359807195Uncharacterized proteinMembrane fatty acid translocation>2
LOC100798112
gi|356539666Cinnamoyl-CoA reductase 1Cell wall biosynthesis>2>2
gi|351724869Uncharacterized proteinContains a trypsing inhibitor>2>21-21-21-2
LOC100306662domain
gi|356505888RuBisCO-associated protein-likePhotosynthesis>2>2>2>2
gi|57152459725.3 kDa vesicle transport protein-Boron intake>2
like
gi|351723027Stearoyl-ACP desaturase 2Fatty acid biosynthesis>21-2
gi|571470673Peroxisomal (S)-2-hydroxy-acidPhotorespiration>2>20.5-1 >2
oxidase GLO1
gi|358248468Uncharacterized proteinEpimerase domain, sugar>2>2
LOC100787970metabolism
gi|356526314Coatomer subunit beta-1-likeIntracellular trafficking0.5-1
gi|571476759Sucrose synthase-like isoform X2Carbohydrate metabolism>2
gi|35651618660S ribosomal protein L9-likeProtein synthesis>2>2
gi|351723535Uncharacterized proteinSimilar Ribosomal_L16; Protein1-2
LOC100305619synthesis
gi|35654972250S ribosomal protein L3-2,Protein synthesis1-2>2
chloroplastic-like
gi|571568318Exocyst complex componentCell growth>2
SEC5A-like
gi|356555176Protein PAT1 homolog 1-likeMitotic chromosome segregation>2
gi|351722913Protein disulfide isomerase-likeProtein synthesis>21-2
precursor
gi|356548745Trafficking protein particleUnknown>2
complex
gi|356563759L-arabinokinase-like isoform 1Cell wall biosynthesis>21-2 1-2
gi|356562473Xylose isomerase [Glycine max]Cell wall biosynthesis>2>2>20.5-1 1-2
gi|356563694ATP synthase protein MI25-likeMembrane bound proton transport>2>21-2
gi|351723615Aspartate aminotransferaseCarbon and nitrogen metabolism1-21-2>2>2>2>21-2
glyoxysomal isozyme AAT1
precursor
gi|571495263Protein disulfide-isomerase 5-4-Protein folding>2
like
gi|358248540Uncharacterized proteinSieve tube occlusion>2>21-2
LOC100778245
gi|358248512Uncharacterized proteinDomain involved in fatty acid1-2>21-2>2
LOC100796978biosynthesis
gi|356534817Exocyst complex componentVesicle traffiking>21-2
EXO70A1-like
gi|356543530UDP-D-apiose/UDP-D-xyloseCell wall integrity>2
synthase 2-like
gi|35980726560S ribosomal protein L35aProtein Synthesis>21-2
gi|356531794V-type proton ATPase subunit d2-Metabolism>2>2
like isoform 1
gi|571517750Aminomethyltransferase,Metabolism1-2>21-2 1-21-2
mitochondrial-like
gi|359807469Molecule involved in rac1 cellCell cycle regulation>21-21-2
signaling
gi|356555809GlycosyltransferaseGrowth Factor modulation>2
gi|356538962Protein phosphatase methylesteraseRegulates phosphatase-2>2
1
gi|351727711Probable 60S ribosomal proteinProtein Synthesis>2
L27a
gi|356558075Cytosolic triosephosphateGlycolosis, Starch Synthesis>2>21-2
isomeraseModulation
gi|356548593Ribosome biogenesis regulatoryProtein Synthesis>2
protein
gi|356531391Probable beta-D-xylosidase 6Cell wall biosynthesis>2
gi|571465945Mitochondrial ATP synthaseRespiration>2
subunit delta
gi|356555162Ribosomal L1 domain-containingProtein Synthesis1-20.5-1
protein 1
gi|356525010V-type proton ATPase subunit FRespiration1-21-2
gi|571445972Pre-mRNA-processing factor 39-RNA export Protein Synthesis1-2
like isoform X3
gi|356570229Elongation factor 1-alpha isoformCell growth and differentiation1-21-21-2
1
gi|356500305Probable subtilisin-like serineCell Cycle, PCD1-21-2>21-2
protease
gi|356531651Chloriplastic 50S ribosomalProtein Synthesis1-2
protein L1
gi|356568070Multiple inositol polyphosphateSeed Development1-2
phosphatase
gi|356575355CalreticulinCalcium ion signaling and protein1-21-2
folding
gi|356523720Mitochondrial-processingRespiration and mitochondiral1-2
peptidase subunit alpha-likegrowth
gi|358249364Uncharacterized proteinPhloem development1-21-2 1-2
LOC100812318
gi|3565321092,3-bisphosphoglycerate-Glycolysis1-21-2
independent phosphoglycerate
gi|356525776SC35-like ProteinPre-mRNA Splicing0.5-1 1-2
gi|351726214calreticulin-1 precursorCalcium ion signaling and protein0.5-1 1-21-2
folding
gi|359807453Ribulose bisphosphate carboxylaseCalvin cycle1-2 1-21-2
small chain
gi|356511113Anthocyanidin 3-O-Glucose Transfer1-2
glucosyltransferase 5-like
gi|571560814Conserved oligomeric GolgiIntracellular trafficking1-21-21-21-21-21-2
complex subunit 4-like isoform X2
gi|571490167Uncharacterized proteinCellulose biosynthesis1-21-2
LOC100816765 isoform X1
gi|571563805Glucosidase 2 subunit beta-likeCarbohydrate metabolism1-21-21-2
gi|356531894Isopentenyl-diphosphate Delta-Isoprenoid biosynthesis1-2 1-2
isomerase II
gi|356552396Galactinol-sucroseUpregulated during seed filling0.5-1 0.5-10.5-1
galactosyltransferase
gi|356567472Probable pectate lyase 5-likeExpressed late in pollen0.5-1 0.5-10.5-1
development
gi|571521870Nuclear pore anchor-likemRNA export, telomere0.5-1 0.5-1 0.5-1 0.5-10.5-1
organization
gi|356575418Anthranilate synthase componentAminoacid biosynthesis0.5-10.5-1
I-2
gi|571440773Villin-3-like isoform X4Seed storage protein0.5-1 0.5-1 0.5-1 0.5-1
gi|356575855Beta-conglycinin, beta chain-likeSeed storage protein0.5-1 <0.50.5-1
gi|356531441Uncharacterized proteinUnknown, Nucleic acid binding0.5-1 <0.5
LOC100797009domain
gi|407971020Glycinin G1 precursorProtein storage0.5-1 0.5-1
gi|356516458Staphylococcal nuclease domain-Salt-responsive phosphoprotein0.5-1 0.5-1 <0.5<0.5
containing
gi|35653351760S ribosomal protein L24-likeComponent of ribosomes, protein <0.5 <0.5
synthesis
gi|351721724Uncharacterized proteinSynaptobrevin/VAMP-like <0.5
LOC100306336protein
gi|356501546Dihydrolipoyllysine-residueGlycolytic and Tricarboxylic acid <0.51-2
succinyltransferase component ofpathways
2-oxoglutarate dehydrogenase
complex 2
gi|351726285Uncharacterized proteinContains RNAse E catalytic <0.5 <0.51-2
LOC100305625domain
gi|356506969Coiled-coil domain-containingUnknown <0.5 <0.50.5-1
protein 124-like
gi|351723585HMG I/Y like proteinNitrogen metabolism0.5-1 0.5-1
gi|525507423Allergen Gly m Bd 28K precursorProtein storage in cotyledons<0.5<0.5
gi|356509275Glutamate synthase [NADH],Ammonium derived reserves0.5-1 <0.5 <0.5 <0.50.5-1 0.5-10.5-1
amyloplastic-likemobilization
Resistance against abiotic stressRatio Treatment/Control
Accession numberGene nameFunctionSYM00057BSYM00596SYM00052SYM00002SYM00046SYM00218SYM00508SYM00940
gi|356532293Plastid-lipid-associated prot.,Enhanced development under>21-2
chloroplastic-likestress
gi|358249004Uncharacterized proteinCutA domain involved in copper>2>2>2
LOC100792337tolerance
gi|351727160Uncharacterized proteinAntioxidant in organeles exposed>2>2
LOC100306620to ROS
gi|351726968Uncharacterized proteinHeat-Shock Protein domain1-2>2
LOC100500475
gi|351723027Stearoyl-ACP desaturase 2Upregulated in cold temperature>21-2
gi|351725976Heat shock protein 90-2Stress response 1-21-2
gi|571466979Polyphenol oxidase A1,Wounding response, disease1-2>2>2 1-21-2
chloroplasticresistance
gi|351727799Uncharacterized proteinPathogenesis-related BetvI>2
LOC100305838domain
gi|356564319Thioredoxin H-typeDefense against oxidative damage>20.5-11-2
gi|351727843Seed linoleate 9S-lipoxygenase-3Pest resistance and response to1-2 1-20.5-10.5-1 0.5-1
wounding
gi|571528717Glutathione reductase, cytosolicUpregulated under cold stress1-2
isoform X2
gi|359807279Uncharacterized proteinDefense against oxidative damage>2 1-21-2
LOC100811170
gi|35653452440S ribosomal protein S17-likeUpregulated under salt stress1-2>21-2>21-2
gi|356526625Probable importin-7 homologAbiotic stress response>2
gi|356522025Pyrophosphate-fructose6-Nutrient stress>2
phosphate1-phosphotransferase
gi|358248934Uncharacterized proteinStress response 1-2>2
LOC100802108
gi|356526803Probable nucleoredoxin 1-likeDefense against drought/oxidative>2
isoform X1damage
gi|351723089Chalcone-flavonone isomerase 1AUpregulated under drought stress1-2>21-21-20.5-1>2
gi|351723659Probable aldo-keto reductase 1Response to heavy1-2
[Glycine max]metal/herbicide stress
gi|359807506Uncharacterized proteinStress response1-2 1-21-2
LOC100784252
gi|356539794Glutamate decarboxylase 1-likeStress Response>2
gi|571528788UDP-glycosyltransferase 74E2-Stress Response>2
like
gi|356571134Heterogeneous nuclearAbiotic stress response>2
ribonucleoprotein A3
gi|356526320ATP synthase subunit epsilonAbiotic stress (drought) response>21-2
gi|356526019Transmembrane 9 superfamilySalt stress tolerance>2>2
member 4-like
gi|571449076Polyadenylate-binding protein 8Salt stress tolerance>2>21-2
isoform X3
gi|571443884DEAD-box ATP-dependent RNADNA repair under abiotic stress>2>20.5-10.5-1
helicase 8-like
gi|363806992V-type proton ATPase catalyticStress response>2>21-2
subunit A-like
gi|351720981Glucan endo-1,3-beta-glucosidaseUpregulated under nematode>21-2 1-21-2
precursorstress
gi|571493372Anamorsin homologDefense against oxidative damage1-2
gi|359807560Mitochondrial outer membraneRespiration, defense against1-21-21-2
proteinoxidative dmg
gi|351724085Glutathione S-transferase familyDefense against oxidative damage1-21-2 1-21-2
protein
gi|363814589Peroxidase precursorPlant defense1-2 1-2
gi|356539207Apoptosis-inducing factorUpregulated under flood stress 1-21-21-2
homolog A-like
gi|358249134Uncharacterized proteinDefense against oxidative damage 1-2
LOC100782451
gi|356576221Aquaporin PIP2-1-likeWater stress 1-2
gi|351720940Glutathione peroxidase familyDefense against oxidative damage1-2
gi|356507345Chaperone protein ClpB4,Heat shock resistance0.5-11-21-20.5-1
mitochondrial-like
gi|35651857126S protease regulatory subunitChaperone and ATPase subunit of1-21-21-2
10Bprotease
gi|35824899083 kDa Heat Shock ProteinStress response1-2
gi|351734408Glutaredoxin (GRX) family similarRedox and defense against1-21-21-2
to humanoxidative dmg
gi|571501181Proton pump-interactor 1-likeStress response>21-2 1-21-2
isoform X2
gi|351723857Peroxisomal betaine-aldehydeSalt tolerance>2>21-21-21-2
dehydrogenase
gi|356519792Flavonoid 3′-monooxygenaseAbiotic stress (drought) response>2 1-2
gi|351721446uncharacterized proteinDefense against oxidative damage>2>2>2
LOC100499951
gi|351722971Phytochrome ARegulates isoflavone levels>2>2
gi|358249004Uncharacterized proteinDivalent ion tolerance protein>2>2>2
LOC100792337
gi|358248196Polygalacturonase inhibitor 1-likeInhibit polygalacturonase1-2>2>2>2
precursor
gi|356550171Beta-galactosidase 8-like isoform 1Lipid degradation1-21-21-2
gi|356552408Transmembrane 9 superfamilyCopper balance>21-2
member 4-like
gi|356556396Aldehyde dehydrogenase family 7Upregulated under salt stress>2
member
gi|356505665S-adenosylmethionine synthase 1-Resistance to abiotic stress1-21-2
like isoform 1
gi|356563248FAM10 family protein At4g22670-Heat shock resistance1-2
like
gi|356539060Vacuolar-sorting receptor 1-likeIntracellular trafficking>21-2
gi|356536151Alpha-L-fucosidase 2-likeUpregulated under stress1-2 1-21-21-21-2 1-21-21-2
gi|359807261Soyasaponin IIIGeneral stress response1-21-21-2 1-21-21-2
rhamnosyltransferase
gi|356575387Prolyl endopeptidase-likeAbiotic stress (drought) response1-21-21-21-20.5-11-21-2
gi|358249064Uncharacterized proteinGeneral abiotic resistance>2 1-21-21-2 1-21-21-2
LOC100795412
gi|571439125Glutathione reductase, cytosolic-Oxidative stress response 1-21-21-2
like
gi|35655937626S protease regulatory subunit 7-Innate Immunity of plants1-21-21-21-21-21-2
like
gi|571517750Aminomethyltransferase,Breakdown of drought stress1-2>2 1-21-21-2
mitochondrial-likemetabolite
gi|356569099Uncharacterized proteinDefense against oxidative damage1-2 1-21-21-2
LOC100816075
gi|351726848Seed linoleate 9S-lipoxygenaseGeneral stress response1-2 1-20.5-1 1-21-2
gi|571433220Uncharacterized proteinDessication stress response1-21-2
LOC100305891
gi|571480718Vacuolar (H+)-ATPase G subunitPhotosynthesis1-2
gi|356572832MEthionine adenosyltransferaceAdoMet pathway, Growth1-21-2
gi|356564135Probable subtilisin-like serineCell Cycle, PCD1-21-2
protease
gi|351727803uncharacterized proteinRiboflavin production 1-21-2
gi|356556866Probable linoleate 9S-Root lateralization and defense 1-21-21-2
lipoxygenase-4
gi|356517182S-norcoclaurine synthaseSecondary metabolite production1-2 1-21-21-2
gi|356563300Probable beta-fructofuranosidaseGrowth and wound response1-21-21-21-21-2
gi|351727703Dihydroflavonol reductaseFlavanoid production/Stress1-2
Response
gi|351722222LipoxygenaseDefense against oxidative damage1-2 1-21-2
gi|351721496Copper amine oxidase precursorwound/pathogen response1-2>20.5-1 1-2
gi|571497607Probable chloroplastic 20 kDaHeat-shock and stress response <0.5 1-21-2
chaperonin
gi|356557126Vicianin hydrolasePlant defense 1-21-2
gi|571445503Villin-2-like proteinStructure 1-20.5-1 1-2
gi|35655156818.2 kDa class I heat shock proteinStress response0.5-10.5-1
gi|356497361Chaicone SynthasePlant defense, antimicrobial0.5-10.5-1
gi|356554094DEAD-box ATP-dependent RNASalt stress adaptation0.5-1
helicase 53
gi|351727184Seed maturation protein PM30seed maturation protein PM24,0.5-1
Drought tollerance
gi|356575853Beta-conglycininSeed storage protein<0.5
gi|351723125Glutathione S-transferase GST 24ROS response and>21-2>20.5-11-20.5-1
Dextoxification
gi|571455778Probable glutathione S-transferaseROS response and0.5-10.5-1
Dextoxification
gi|351722245Seed maturation protein PM3Heat shock protein, seed0.5-10.5-1
maturation
gi|571453722Programmed cell death protein 4Abiotic stress response>2>20.5-10.5-1
gi|571481365Late embryogenesis abundantDehydration tolerance, seed0.5-1 1-21-20.5-10.5-1 <0.5
protein D-34formation
gi|351724511Beta-conglycinin alpha primeSeed storage protein<0.5<0.5
subunit precursor
gi|363814316ATP synthase (C/AC39) subunitRespiration0.5-10.5-1
gi|359807588NADP-Binding oxidoreductaseRespiration0.5-10.5-1
gi|571480648granule bound starch synthase IaCarbohydrate Storage0.5-1 0.5-10.5-1 0.5-1
gi|351722615seed maturation protein PM24seed maturation protein PM240.5-10.5-1
gi|571463841PR 10-like proteinRNA degredation0.5-1
gi|57147262160S ribosomal protein L7-4protein synthesis0.5-1
gi|571513400Amyloplastic glutamate synthaseCarbohydrate Storage0.5-1
[NADH]
gi|351722277P24 oleosinHelps form oil barter to protect0.5-1
seeds
gi|35172318360S ribosomal protein L6Protein synthesis0.5-10.5-1
gi|571460036protein disulfide-isomeraseEndosperm and seed development0.5-10.5-1
gi|91214131ribosomal protein S4Protein synthesis0.5-1
gi|351725291Ca+2-binding EF hand proteinCell signalling and regulation0.5-1<0.50.5-1
gi|571445521Galactinol-sucroseCarbohydrate metabolism0.5-1 0.5-1<0.5
galactosyltransferase 2
gi|356506190Transketolase, chloroplasticEnergy metabolism0.5-1 0.5-10.5-1 0.5-1 0.5-1 0.5-1
gi|571560306Cysteine synthase-like isoform X3Amino acid downregulated0.5-1 0.5-1
protein.
gi|571493751Squamous cell carcinoma antigenPossible alarm signal that stresses1-20.5-1 0.5-1
the plant
gi|351727923LEA protein precursorGeneral abiotic resistance0.5-1 0.5-1<0.50.5-1
gi|351726932Uncharacterized proteinPathogenesis-related BetvI <0.5 <0.5 1-21-2
LOC100305867domain
gi|356509275Glutamate synthase [NADH],Amino acid downregulated0.5-1 <0.5 <0.5 <0.50.5-10.5-1 0.5-1
amyloplastic-likeprotein
gi|57144363935 kDa seed maturation proteinUnknown, associated with <0.50.5-1<0.5<0.5
drought stress
gi|351724439Seed biotin-containing proteinSeed maturation protein <0.5 <0.50.5-1<0.5
SBP65
gi|356533407Embryonic protein DC-8-likeSeed maturation protein <0.5 <0.50.5-1<0.50.5-1
gi|571477629Low-temperature-induced 65 kDaSeed maturation protein0.5-1 <0.50.5-1<0.50.5-1
prot.
gi|351720785Glycinin precursorPrecursor of seed storage protein0.5-1 0.5-1
gi|571481071Nucleolar protein 14-likeProtein synthesis0.5-1 0.5-1
gi|356506138la-related protein 6-likeThermal stress response0.5-1
gi|356509908Cysteine proteinase 15A-likeStress response0.5-1
gi|356505489NPL4-like protein 1-likeProtein degradation0.5-1
gi|356512347Signal recognition particle subunitTargeting secretory proteins to0.5-1 0.5-1 0.5-1
SRP72-likeER
gi|351722383GlucosyltransferaseMolecular acclimation to stress0.5-1 1-21-20.5-1
gi|35172607851 kDa seed maturation proteinUpregulated during seed drying <0.50.5-1 0.5-1
precursor
gi|351727923Lea protein precursorUpregulated during seed drying0.5-1 0.5-1<0.50.5-1
gi|356531617Ubiquitin carboxyl-terminalMolecular acclimation to stress0.5-1
hydrolase 24-like
gi|356516170Nascent polypeptide-associatedMolecular acclimation to stress<0.5
complex subunit alpha-like protein
2-like
gi|356577005Enolase-phosphatase E1-likeMethionine salvage<0.5
gi|571482993Enolase-phosphatase E1-likeMethionine salvage<0.5
gi|351723669Em proteinWater stress response <0.5<0.5
gi|351722444maturation protein pPM32Stress response <0.5 <0.50.5-1
[Glycine max]
gi|356561627Low-temperature-induced 65 kDaWater stress response0.5-10.5-1
protein-like
gi|356542936Riboflavin synthase-likeStress response0.5-1 0.5-10.5-1
gi|356535993Beta-conglycinin, alpha chainSeed storage protein<0.5<0.5<0.5
gi|356548291Ethylene-insensitive proteinStress response <0.5
gi|356516335Carbamoyl-phosphate synthaseAMF nitrogen and phosphorus>2
small chainmetabolism
Symbiosis enhancementRatio Treatment/Control
Accession numberGene nameFunctionSYM00057BSYM00596SYM00052SYM00002SYM00046SYM00218SYM00508SYM00940
gi|380848783Lectin precursorUpregulated under Aspergillus>2>2
infection
gi|356555176Protein PAT1 homolog 1-likeInvolved in plant-microbe>2
interaction
gi|571467278Caffeoyl-CoA O-Plant defense1-2
methyltransferase-like
gi|356535145Benzyl alcohol O-Synthesis of anti-pathogen1-21-2
benzoyltransferase-likecompounds
gi|356554704UDP-glycosyltransferase 85A2-Synthesis of anti-pathogen>2
likecompounds
gi|356539170Beta-adaptin-like protein C-likeAnti-fungal infection>2
gi|571470673Peroxisomal (S)-2-hydroxy-acidPlant disease resistance>2>20.5-1 >2
oxidase GLO1
gi|356495574Heat shock protein 90-1-likePlant disease resistance1-2
gi|571465187WD repeat-containing protein 74-Rhizobium induced cell growth>2
like
gi|356577969Isoflavone reductase-likeNod gene induction1-21-21-2
gi|571436840Pectinesterase/pectinesterasePlant defense1-21-2>21-21-2
inhibitor 18-like
gi|356538839Allene oxide synthase,Defense response to mechanical1-21-21-21-2
chloroplastic-likestress
gi|356508869Lysosomal alpha-mannosidase-likeBacterial colonization of lytic1-21-21-2
compartment
gi|356548301Phenylalanine ammonia-lyase classUpregulation after wounding and1-21-2
2-likeinfection
gi|358249162Uncharacterized proteinDefense response to1-21-21-2
LOC100780100Pseudocercospora
gi|356553956Oxygen-evolving enhancer proteinDefense response to bacteria1-21-2
2-2
gi|356568935Uncharacterized proteinNitrogen fixation1-2
LOC100817481
gi|356541838Methionine S-methyltransferaseSupports growth of1-21-2
Corynebacterium
gi|571496006Gamma-glutamyl hydrolaseSymbiosis signal for1-21-21-21-2
Bradyrhizobium
gi|571460508Exosome complex exonucleaseActivated under conditional0.5-1 0.5-1 0.5-1
RRP44-likesymbiosis
gi|356556999Ubiquitin-NEDD8-like proteinSymbiosis: Auxin release1-21-2
RUB1-like
gi|571472984ras GTPase-activating protein-Possible symbiosis by G-protein <0.51-2
binding proteinbinding
gi|356554746H/ACA ribonucleoprotein complexSymbiosis signal for0.5-10.5-1 0.5-1
subunit 4Bradyrhizobium
gi|356508718Ras-group-related LRR protein 9-Cell to cell signalling<0.50.5-1
like
gi|356565179Leghemoglobin reductase-likeFerric leghemoglobin reduction1-2
Growth Promotion
Proteins involved in the breakdown of seed stored reserves and playing important roles in the stimulation of continued growth during germination were up-regulated by endophytes. This class of proteins includes beta-fructofuranosidases, fructan 1-exohydrolases and carboxypeptidases involved in the mobilization of sucrose, fructans and insoluble proteins respectively, for the release of glucose, fructose and amino acids (Fincher 1989, Annu. Rev. Plant Physiol. Plant Mol. Biol. 40:305-46;). Those results show that bacterial endophytes induce a faster release of nutrients from the seed, leading to augmented growth at early stage of plant development. The levels of proteins playing a role in cell proliferation and elongation were also increased in endophyte-inoculated seedlings. This class of proteins includes dynamins, histones, a ribonucleoside-diphosphate reductase, pectinesterases and villins, involved in cell division, chromatin structure, DNA synthesis, cell wall remodeling and elongation respectively (Hepler et al. 2001, Annu. Rev. Cell Dev. Biol. 17:159-87, Kang et al. 2003, The Plant Cell 15(4): 899-913, and Imoto et al. 2005, Plant Mol. Biol. 58:177-192). Those results demonstrate that, in response to the endophytic bacteria tested, the two types of plant growth, proliferation and elongation, are promoted, leading to substantial growth enhancement.
Resistance Against Stress
A number of proteins involved in resistance against stress were significantly up-regulated in wheat under stress induction and the presence of endophytes. The level of several proteins playing a role in resistance against oxidative stress by scavenging reactive oxygen species was higher in inoculated plants including glutathione S-transferases (GST), peroxidase and ascorbate oxidase (Apel and Hirt 2004, Annu. Rev. Plant Biol. 55:373-99). Those results shows that in addition to plant growth, the endophytes tested promoted the general pathways involved in resistance against oxidative stress. The proteomics data-set also revealed the strong induction of a pectinesterase by SYM00011 and SYM00057B in wheat that might play a role in drought resistance as previously described (Mays et al. WO2013122473).
Symbiosis Enhancement
In maize under normal conditions, only GST was up-regulated, while other abscisic acid (ABA) and stress inducible proteins were down-regulated. The down-regulation of ABA and stress inducible proteins in maize was positively correlated with the down-regulation of proteins associated to programmed cell death, pathogen resistance and hypersensitive response. Moreover, the replication factor C, subunit 3 that negatively regulates plant defense was significantly overexpressed in the SYM00057b inoculated maize seedlings. Those results are consistent with the conventional wisdom that, under normal condition, the establishment of symbioses with beneficial microbes involves decrease in the expression of genes associated to the plant defense system (Samac and Graham, 2007, Plant Physiol. 144(2):582-587).
In addition, several proteins directly associated with beneficial symbioses are up-regulated in the wheat and maize. Several of these proteins are homologous to proteins involved in nodule formation in legumes. Many genes involved in nodulation, such as nodulation receptor kinases are broadly distributed in the plant kingdom, even in plants incapable of forming nodules, as is the case of maize (Endre et al. 2002, Nature 417:962-966). Some of these conserved receptors may sense bacterial signals in symbiotic associations other than Legume-Rhizobia and this may explain why the nodulation factors from Badyrhizobium japonicum are able to enhance seed germination and root growth in maize (Souleimanov et al. 2002, J. Exp. Bot. 53(396):1929-1934).
Results for Soybean Plants
In one embodiment, synthetic combinations of soybean plants and bacterial endophytes (e.g. SYM00057B, SYM00596, SYM00052, SYM00002, SYM00046, SYM00218, SYM00508 and SYM00940) grown under normal conditions produce a proteomic signature including polypeptides associated with growth promotion, resistance against stress and mechanisms involved in symbiosis enhancement (Table 18).
In particular, one or more pathways including the biosynthesis of proline, cell walls, methionine, carbohydrates, proteins, isoprenoid and flavonoids are modulated, e.g., at least one member (e.g., one, two, three, four, five, or six proteins) of a pathway such as arginase, xylose isomerase, sucrose synthase, 60S ribosomal protein L9, isopentenyl-diphosphate delta-isomerase II and flavonoid 3′-monooxygenase are increased relative to a reference soybean plant.
One or more proteins (e.g., one, two, three, four, five, six, seven, eight, nine, or ten proteins) that increase during seed filling, maturation and drying are also modulated, e.g., villin, beta-conglycinin, seed maturation proteins PM24 and PM30, Late embryogenesis abundant protein D-34, seed biotin-containing protein SBP65, embryonic protein DC-8-like, 35 kDa and 51 kDa seed maturation proteins and LEA protein precursor are decreased relative to a reference soybean plant.
One or more proteins (e.g., one, two, three, four, five, six, seven, eight, nine, or ten proteins) with demonstrated effects in plant defense or tolerance against biotic and abiotic stresses are modulated, e.g., 4-coumarate—CoA ligase, thioredoxin, gluthathione S-transferase, glutathione reductase, peroxidase, peroxisomal betaine-aldehyde dehydrogenase, lipoxygenase, heat shock protein 83-like, class I heat shock protein, and mitogen-activated protein kinase are increased relative to a reference soybean plant. In particular, proteins associated with drought tolerance, e.g., plastid-lipid-associated protein, ATP synthase subunit epsilon, chalcone-flavone isomerase, flavonoid 3′monooxygenase, aquaporin PIP2-1-like and prolyl endopeptidase-like are increased relative to a reference soybean plant.
One or more proteins (e.g., one, two, three, four, or five proteins) involved in stress response in the form of apoptosis, plant cell death or cellular degradation, e.g., programmed cell death protein 4, PR10-like protein, squamous cell carcinoma antigen, ubiquitin carboxyl-terminal hydrolase 24-like, and Ethylene-insensitive protein are decreased relative to a reference soybean plant.
One or more proteins (e.g., one, two, three, four, or five proteins) involved in the establishment of symbiosis with beneficial microbes and/or defense against pathogenic microbes, e.g., lectin, isoflavone-reductase-like, pectinesterase inhibitor 18-like, methionine S-methyltransferase and gamma-glutamyl hydrolase are increased relative to a reference soybean plant.
One or more proteins (e.g., one or two proteins) involved in symbiosis with root nodule forming microbes, a category different to the bacterial endophytes used in the experiment, e.g., H/ACA ribonucleoprotein complex and Leghemoglobin reductase-like, are decreased relative to a reference soybean plant.
Three proteins common to most synthetic combinations of soybean plants with endophytes that showed increased levels relative to a reference soybean plant are: alpha-L-fucosidase 2-like, chalcone-flavone isomerase and 26S protease regulatory subunit 7-like.
Three proteins common to most synthetic combinations of soybean plants with endophytes that showed decreased levels relative to a reference soybean plant are: seed biotin-containing protein SBP65, 35 kDa maturation protein and transketolase.
In another embodiment, proteins in the amino acid metabolism pathways such as aspartate aminotransferase glyoxysomal isozyme AAT1 precursor are increased relative to a reference soybean plant whereas glutamate synthase amyloplastic-like isoform X1 is decreased relative to the reference plant.
