Identification of Soybean Proteins and Genes Differentially Regulated

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Identification of Soybean Proteins and Genes Differentially Regulated in Near Isogenic Lines Differing in Resistance to Aphid Infestation Laurent Brechenmacher, Tran Hong Nha Nguyen, Ning Zhang, Tae-Hwan Jun, Dong Xu, M.A. Rouf Mian, and Gary Stacey J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00146 • Publication Date (Web): 09 Sep 2015 Downloaded from http://pubs.acs.org on September 9, 2015

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Journal of Proteome Research

Identification of Soybean Proteins and Genes Differentially Regulated in Near Isogenic Lines Differing in Resistance to Aphid Infestation Laurent Brechenmacher1, Tran Hong Nha Nguyen1, Ning Zhang2, Tae-Hwan Jun3,^, Dong Xu2, M. A. Rouf Mian3,+, Gary Stacey1,*

1

Divisions of Biochemistry and Plant Sciences, National Center for Soybean

Biotechnology, University of Missouri, Life Sciences Center, Columbia, MO 65211, USA 2

Computer Science Department and Christopher S. Bond Life Sciences Center,

University of Missouri, Columbia, Missouri 65211 3

USDA-ARS, Department of Horticulture and Crop Science, The Ohio State University,

1680 Madison Avenue, Wooster, OH 44691USA

*

Corresponding author: Prof. Gary Stacey,

271E Christopher S. Bond Life Sciences Center University of Missouri Columbia, MO 65211 Office phone: 573-884-4752 Fax number: 573-884-9676 Email: [email protected]

1 ACS Paragon Plus Environment


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Running title: Genes and proteins regulated during soybean-aphid interaction

The abbreviations used are: NIL, Near Isogenic Line; RNAseq, RNA sequencing; NBSLRR, Nucleotide-Binding Site Leucine-Rich Repeat


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ABSTRACT Soybean aphid, is an important pest causing significant yield losses. The Rag2 locus confers resistance to soybean aphid biotypes 1 and 2. Transcriptomic and proteomic analyses were done over a 48 h period after aphid infestation using near isogenic lines (NILs) differing at the Rag2 locus. Comparing the Rag2 and/or rag2 lines identified 3445 proteins with 396 differentially regulated between the two lines, including proteins involved in cell wall metabolism, carbohydrate metabolism, and stress response. RNAseq transcriptomic analysis identified 2361 genes significantly regulated between the resistant and susceptible lines. Genes up-regulated in the Rag2 line were annotated as involved in cell wall, secondary and hormone metabolism, as well as in stress, signaling and transcriptional responses. Genes down-regulated in the Rag2 line were annotated as involved in photosynthesis and carbon metabolism. Interestingly, two genes (unknown and mitochondrial protease) located within the defined Rag2 locus were expressed significantly higher in the resistant genotype. The expression of a putative NBS-LRR resistant gene within the Rag2 locus was not different between the two soybean lines, but a second NBL-LRR gene located just at the border of the defined Rag2 locus was. Therefore, this gene may be a candidate R gene controlling aphid resistance.

Keywords: Soybean, Aphid, Transcriptomic, Proteomic, NIL, Resistant, Susceptible



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INTRODUCTION Soybean aphid (Aphis glycines Matsumura) is an insect belonging to the arthropod phylum which has recently become a very important pest of soybean in North America. The soybean aphid is an invasive species originating from eastern Asia and was first detected in the USA in 20001. Aphids spread quickly and were detected in 30 different states by 20091. This rapid spread is due to the ability of this insect to travel long distance in its winged form (alate) and the widespread presence in USA of its overwintering host Rhamnus cathartica (common Buckthorn)2. Eggs deposited on buckthorn hatch in March-April into wingless aphids (apteran) which then reproduce parthogenically for 2-3 generations. They differentiate into alate aphids which migrate in May-June from buckthorn to soybean, its main secondary host3. The high fecundity potential and the short development period of aphids enable rapid clonal multiplication on soybean (up to 15 generations) during the growing season. Under optimal conditions, the aphid population can double in 1.5 days. However, doubling time in the field usually takes longer and was estimated to be 6.8 days4. In autumn, aphids differentiate into winged males and females, which fly back to buckthorn where the females, after sexual reproduction, lay eggs that will hatch during the next spring season. The economic loss for the soybean industry due to the presence of aphid was estimated to be about 4 billion dollars annually3. Aphids use their stylet to reach, mainly intercellularly, the sieve element to ingest phloem sap. Although this type of feeding does not wound the plant as badly as chewing insects, the infestation of a single soybean plant by thousands of aphids can divert enough photosynthate to affect


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soybean development. Furthermore, soybean aphids are also competent vectors to transmit Soybean or Alfalfa mosaic virus5. Aphids also secrete honeydew, which can promote fungal growth. The growth of saprophytic fungi on soybean leaves hinders stomatal function and prevents efficient light capture, reducing photosynthesis. Collectively, perhaps through a variety of mechanisms, aphid infestation can decrease soybean yield (up to 40%). Typical symptoms of aphid infestation include shorter plants with yellow leaves, with fewer pods containing fewer and smaller seeds with less oil than healthy soybeans6. Different management systems have been developed to control soybean aphid infestation. These include the use of insecticides, including pyrethroid and organophosphate types, with the recommendation that these be applied when the economic threshold of 250 aphids per plant is reached4. Although proper use of these insecticides can greatly reduce the damaging effects of aphids on soybean yield, this approach is costly (~33 $/hectare), is detrimental for the environment, can lead to the development of insecticide resistant aphids and also adversely affects the population of insects that normally prey on aphids4. Biological control of aphids using predators is another method to control aphid multiplication and is based on the idea that aphids rarely reach the status of pest in Asia due to the presence of effective predators. Efficient predators of aphids present in USA include insects, such as the insidious flower bug (Orius insidiosus) or those belonging to the Coccinellidae (lady beetle) family1. The use of soybean lines naturally resistant to aphids is another management approach to control soybean aphids. Resistance mechanisms to insects are usually



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classified in 3 categories including antibiosis, antixenosis and tolerance, depending on how plants can modify the development and behavior of the insect. Antibiosis resistance affects the biology of the insect (increased mortality, reduced fecundity) while antixenosis resistance affects the behavior of the insect (non-preference). Tolerance is another process when the plant is able to withstand the presence of the insect without significant impact on the host biology or behavior. Past surveys of the soybean germplasm collection identified soybean lines that exhibited resistance to Aphis glycines. Genetic analysis of these lines identified five resistance (Rag, resistance to Aphis glycines) loci, including Rag1, Rag2, Rag3, Rag4 and Rag53. Of these, Rag1 and Rag2 are the best characterized. The single dominant locus, Rag2, providing resistance to biotypes (see below) 1 and 2 (based on soybean cultivar resistance patterns) of the soybean aphid, was mapped on soybean chromosome 13 from Plant Introduction (PI) 243540. Further genetic mapping of the Rag1 and Rag2 loci identified a 115 kb interval on chromosome 7 and a 54 kb interval on chromosome 13, respectively

7,8

. Both

intervals contain Nucleotide-Binding Site Leucine-Rich Repeat (NBS-LRR) genes (2 for Rag1 and 1 for Rag2), which were proposed as good candidates for aphid resistance genes. Indeed, the tomato Mi1.2 and melon Vat genes, which encode NBS-LRR proteins, were previously shown to confer aphid resistance 9,10. However, at present, the molecular basis for aphid resistance in soybean conferred by the five Rag loci remains undefined. The first commercial soybean aphid resistant cultivar, containing the Rag1 gene, was released in 201011. However, subsequently, aphids were identified that could overcome Rag1 resistance and were termed Biotype 28. Likewise, aphids of biotype 3, able to develop on Rag1 and Rag2 plants, were described12. The appearance of these


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aphid populations that overcome Rag1 and Rag2 resistance underlines the importance of further research to define the molecular basis for soybean aphid resistance with the ultimate goal of breeding plants with durable aphid resistance. Previous studies utilized proteomic analysis of the saliva of diverse aphids (i.e., isolated from pea, Russian wheat, green peach aphids) with the ultimate goal of identifying effector proteins that are injected into the plant to enhance susceptibility13-16. Presumably it is these effector proteins that are recognized by the plant to induce resistance. Protein accumulation and gene expression were also analyzed to determine the effect of photoperiod on the reproductive mode of the pea aphid17, for which a genomic sequence is available18. A number of studies have also examined plant gene expression in response to aphid infection, including studies involving soybean/ soybean aphid19,20,21, 25,26

Arabidopsis/green

,cabbage/cabbage aphids

peach

aphids22-24,

Arabidopsis/cabbage

aphids

27

, mustard/cabbage aphids28, celery/green peach aphids

22

, sorghum/greenbug aphids22, tobacco/tobacco aphid22, apple/rosy apple aphids22,

barley/bird cherry oat aphids29, wheat/wheat aphids30 or barley/Russian wheat aphids31. These studies resulted in the identification of hundreds of aphid responsive genes, which are mainly involved in photosynthesis, cell wall modification, oxidative stress, hormonal regulation, signaling or transcriptional regulation. However, in contrast to these numerous transcriptome studies, only a single publication, using wheat and cereal aphids

32

, utilized proteomic approaches to examine the plant response to aphid

infection. In this study, we used both transcriptomic and proteomic approaches to examine the soybean response to aphid infestation over a 48 hour time course. In order to define



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those responses specifically associated with resistance, we compared two soybean near isogenic lines (NILs) that differed at the Rag2 locus. The data obtained reveal a significant number of mRNA and protein changes between these two lines, including the differential expression of genes located in or near the Rag2 locus. EXPERIMENTAL SECTION Procedures and Reagents: Development of NILs The homozygous aphid resistant (Rag2/Rag2) and aphid susceptible (rag2/rag2) NILs were developed by backcrossing the Rag2 gene from its original source, PI 243540, into soybean aphid susceptible (rag2) cultivar Wyandot (the recurrent parent). Wyandot is an F4-derived selection from a three-parent cross (Northrup King S29-18 × PI 274421) × Ohio FG1. Ohio FG1 was released by The Ohio State University and Ohio Agricultural Research and Development Center (OARDC) as a food-grade soybean33. A total of four backcrosses were used to develop the BC4F1 seeds. The F1 seedlings developed from the first cross and each backcross generation were screened for resistance to the soybean aphid biotype 2 in the greenhouse according to Mian et al.34. A BC4F2 plant segregating for resistance to the soybean aphid was identified by marker assisted selection and seeds from homozygous resistant (Rag2/Rag2) and homozygous susceptible (rag2/rag2) BC4F2:3 plants originating from the segregating BC4F2 plant were harvested as NILs.

Soybean tissue collection


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The seedlings of the NILs were grown in an environment controlled greenhouse. The experimental design was a randomized complete block with four replications. For each NIL, four seeds per replicate for each of the four sampling times (0, 8, 24, 48 hrs after aphid infestation) were planted in 4-cm diameter x 15-cm deep plastic conetainers in a USDA greenhouse at Ohio Agricultural Research and Development Center (OARDC), Wooster, OH and the best two seedlings per conetainer were kept after germination. The conetainers were arranged in a rack with 8-cm x 12-cm spacing on a greenhouse bench. The greenhouse was maintained at approximately 26/22˚C day/night temperatures with 15 h light daily. The biotype 2 soybean aphids used in this study was from a growth chamber colony maintained at OARDC, Wooster, OH34. At the V2 growth stage, each soybean seedling was manually infested with 15 adult soybean aphids by placing the aphids on the top leaves of the seedling. For the 0 hr samples, just before infestation of seedlings with soybean aphids, the youngest unfolded leaves and the tips of two seedlings representing each replicate of each NIL were collected in a 2 ml tube and were immediately frozen in liquid nitrogen. The samples for 8, 24, and 48 h after infestation, were collected in the same manner, except that the aphids were gently removed from the tissue by using a soft paint brush before tissue collection. The aphids continued to walk away from the resistant seedlings within 12 to 24 hours of placement on the seedlings, so more aphids were added to these seedlings every 12 hrs to have at least 15 aphids per seedling at all times.

Protein and RNA extraction



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Proteins and RNA were extracted from soybean leaves using Trizol reagent (Invitrogen; Carlsbad, CA) according to the manufacturer instruction with the following modifications. Trizol was supplemented with protease inhibitors (Sigma tablet, St Louis, MO, S8820) at a concentration recommended by the provider, before being added to the homogenized frozen tissue. After precipitation of the proteins with isopropanol (0.8 vol per vol of Trizol), pellets were homogenized using a tissue grind pestle (Kontes glass company; Bridgestone, NJ) before being washed 3 times with 0.3 M guanidine hydrochloride in 95% Ethanol. After the final wash, proteins were stored at -80ºC in 100% ethanol until quantification. Protein abundance was estimated using the Quick Start Bradford Dye Reagent and BSA from Bio-Rad (Hercules, CA). Once the last step of the manufacturer’s instructions was reached for RNA isolation, RNA were further purified by adding 0.1 vol of sodium acetate (3M) and 1 vol of chloroform. After a centrifugation at 10000 g for 10 min at 4ºC, RNA present into the aqueous phase were retrieved and precipitated overnight at -20 ºC with 2.5 vol ethanol. The RNA were then washed with 75% ethanol, centrifuged and resuspended in DEPC water. Quantity and integrity of RNA were estimated for each extraction using a NanoDrop (NanoDrop Technologies, Wilmington, DE) and by visual examination after electrophoresis in a 1.2% agarose gel, respectively.

Sample Preparation for MS analysis Fifty µg of total proteins were dissolved into Laemmli buffer, denaturated for 5 min in boiling water and placed on ice. Proteins were then separated by 1D SDS-PAGE using a 10% acrylamide running gel (5 cm) and a 5% acrylamide stacking gel (1.5 cm) until


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the bromophenol blue reach the bottom of the gel. No sample was loaded into two consecutive wells to avoid protein contamination. Gels were stained with Colloidal Coomassie blue. Each gel lane corresponding to one sample were excised and cut into 12 bands of about 0.4 mm. Each band of the gel was minced into about 1 mm3 cube and digested with trypsin as described in

35

with the following modification: gel plugs

were reduced for 30 min at 56ºC with 10 mM DTT in 100 mM ammonium bicarbonate and alkylated for 30 min at room temperature in the dark with 50 mM iodoacetamide in 100 mM ammonium bicarbonate before being rehydrated with 100 µl of trypsin solution (0.02 µg/µl in 50mM ammonium bicarbonate, 10% acetonitrile; Promega, Madison, WI). After trypsin digestion, tryptic peptides were collected as described in35, lyophilized and reconstituded in 10 µl of 1% formic acid. Peptides were centrifuged at 10000 g for 5 min at 4ºC and 8 µl were transferred into MS vial (200046; Sun-SRi,Rockwood, TN) which were stored at 4ºC in the autosampler right before LC MS/MS analysis.

LC MS/MS analysis, protein identification and quantification Tryptic peptides were analyzed on an LTQ Orbitrap XL mass spectrometer operated with Xcalibur (version 2.0.7) and coupled to a nanoflow Proxeon-EasynLC system (Thermo Scientific, Waltham, MA). Tryptic peptides (5µl) were loaded onto a C18 peptide Cap Trap (Michrom Bioresources, Auburn, CA) at a flow of 20 µl/min and a maximum pressure of 200 bars. Peptides were eluted using an 80 min linear gradient from 5 to 45% of solvent B (0.1% formic acid in acetonitrile) at a flow rate of 0.4 µl/min. and separated on fused silica analytical column (150 µM ID x 100 mm) packed with C18 (5 µM, 100 Å; Michrom Bioresources). Solvent A was 0.1% formic acid in MilliQ water.



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Peptides were then electrosprayed using 1.7 kV voltage into the ion transfer tube (180ºC) of the LTQ Orbitrap operating in positive mode. The Orbitrap first performed a full MS scan at a resolution of 30000 FWHM to detect the precursor ion having a m/z comprised between 300 and 2000 and a +2 or higher charge. CID (35% normalized collision energy) was used to fragment the nine most intense precursor ion (1000 counts minimum; parent mass width: plus or minus 0.5 Da) of each full scan which were analyzed by the LTQ linear trap. Exclusion list (mass window: plus or minus 10 ppm) included most common tryptic ions having the following m/z: 421.76, 523.29, 737.71, 761.73, 1106.06, 1142.10, 1150.09. Xcalibur raw data files were imported into Sorcerer (version Version 3.5, Build 4.0.4; Sage-N research, Milipitas, CA) which extracted the peaks and used a Sequest algorithm (version 27) to match the MS and MS/MS spectra to protein sequences36. The database (Glyma 1.0; February 2011) used in this study contains 75778 soybean protein sequences deduced from the soybean genomic sequence and was concatenated with the addition of a decoy sequence, generated by decoy DB creator (www.p3db.org), for each soybean protein to estimate the false discovery rate. Search parameters for tryptic peptides included carbamidomethylation of cysteine and oxidation of methionine as fixed and variable medications, respectively. One miscleavage for trypsin was allowed. Mass tolerances were set at 10 ppm and 1.0 Da for the precursor ion and fragmented ions, respectively. The 12 Xcalibur raw files (12 gel bands) corresponding to proteins extracted from one sample were searched at the same time using Sorcerer to identify at once all the proteins present in a single sample. The results of the different searches were further analyzed using Scaffold (version 3.4.9; Proteome software, Portland, OR) to validate MS and MS/MS data. Only proteins


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identified with at least 2 peptides and a confidence higher than 99% were kept for further analysis. Protein Prophet algorithm was used to assign protein probability37. Peptide Prophet algorithm was used to filter out peptides with mass error higher than 10 ppm38. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. No proteins coming from the decoy database were identified using the search parameter described above. Protein abundance was determined by mass spectral counting and normalized in Scaffold using the quantitative value function. Statistical analysis (t-test) was performed to identify proteins regulated between Rag2 and rag2 lines in response to aphid infestation or not. The cutoff applied to consider that a protein was significantly regulated were a p-Value2 or

Identification of Soybean Proteins and Genes Differentially Regulated

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