Creating VIGS Constructs and Identifying New Candidates

First Rotation Experiences in Steve Whitham’s Lab

By Jesse Walsh

Two Projects: Wet and Dry

Worked under Chunling creating VIGS constructs

Worked under Michelle learning BLAST techniques

Creating VIGS ConstructsChunling had picked out 10 candidate genes

for me to silenceSuccessfully amplified 7 of the 10 using PCR

A7 B2 B8 F5 E12 D9 D10 Verified presence of significant DNA

concentration via Nanodrop

Candidate Genes UsedOld TC New TC EST Clone Annotation

A7 TC225109 TC239091 Gm-c1019-5214  Unknown (Scaffold)

B2 TC218730 TC254732 Gm-c1031-845 GDSL - motif lipase

B8 TC203322 TC257007 Gm-c1040-4326Matrix metalloproteinase MMP2

D9 TC218543 TC238063 Gm-c1072-4342Probable calcium binding protein

D10 TC230385 TC248659 Gm-c1072-4862Receptor lectin protein kinase-like

F5 TC231219 TC253158 Gm-c1076-6736 Unknown (Scaffold)

E12 BQ785921 N/A Gm-c1076-4339 Unknown (Scaffold)

Topo CloneCustomized Topo vector

Codes for a Soybean virus– BPMV RNA2 Contains an ampicillin resistance marker

Ligate the amplified PCR product into the topo vector

TransformationTransform ligated vector to competent E. coli

by heat shockPlate the E. coli ampicillin treated plates and

incubate overnight

Results0 out of the first 36 screens were positive0 out of the 2nd 36 screens were positive1 out of the 3rd 36 screens were positive

Positive colony was for the A7 EST Clone

0 out of the next 72 screens were positive0 out of the final 48 screens were positive

Problems?What am I to conclude?Tested for presence of PCR product, so I know

something is thereE. coli can grow on ampicillin treated plates, so I

know that transformation is happeningScreens produce primer dimer, so I know the reaction

should have been successfulChunling verified that my process and materials were

correctChunling tried screening of plate of D9 D10 E12 and

found 4 positive screens out of 36Am I that unlucky?

Had the screens been successful…If I had been able to obtain more positives by

screening, I would have continued the process of plasmid extraction

MiniPrep kit would have been used to isolate the corresponding plasmids with positive inserts

Confirm the insert with sequencingSoybean plants would then have been

bombarded by the plasmids, and the effects of the gene silencing observed

Meanwhile…I was also learning about BLAST techniques with Michelle

How TC’s are identifiedGenes are being transcribed all the timeIf you were to sequence the DNA in a particular

plant part, at any given time you would find many fragments of RNA being transcribed from the same gene, each of a different length.

If you were to reassemble these fragments, called ESTs, you would be able to reassemble the sequence of the gene.

By taking samples from many individuals of a species, from various parts of the individuals and at different times, you would be able to assemble a consensus sequence of the gene. We call this a TC.

Identification of 1,600 Defense Related Genes:Candidates for VIGS

Criteria #1: homolog of known defense gene in Arabidopsis

1. All soybean Affy consensus sequences were BLASTED (TBLASTX) against the Arabidopsis predicted genes.

2. GO annotation was determined for the top Arabidopsis hit.

3. If any of the GO annotations were related to defense, resistance, wounding or stress the soybean gene was annotated as defense related.

Examples: response to pest, pathogen, parasite or

stress, response to jasmonic acid stimulus, response to pathogenic fungi, systemic acquired resistance

4. Using this approach 1,634 sequences were identified

Criteria #2: Soybean sequences significantly differentially expressed in response to Phakopsora pachyrhizi (Rpp2, van de Mortel et al. 2007).

Criteria #3: Soybean sequences significantly differentially expressed in response to Pseudomonas syringae (Zou et al. 2005).

1,940 sequences identified

3,897 sequences identified

TC100493: Extensin-like protein

‘Untreated’ librariesIrradiated seedling Nitrogen starved seedling Developing stem Leaves and cotyledonsDeveloping leavesPhosphate-starved leavesPhosphate-starved rootsNitrogen-starved rootsRoot hairs and tipsFlowersDeveloping FlowersGerminating seedImmature seed

Defense/symbiosis librariesElicited cell culture Virus-infected leavesPhoma-infected leavesLeaves-C. trifolliAphid-infected shoots Roots-M. incognitaRoots-P. medicaginisRoots-G. versiformeNodules-S. meliloti

22 libraries 115 Total ESTs72 Non-Defense ESTs

ESTs2473111022144175

ESTs21131731113

Criteria 4 and 5: Statistical Analysis of EST data

Example:

LibraryVirus-infected leavesPhoma-infected leavesLeaves-C. trifolliAphid-infected shootsRoots-M. incognitaRoots-P. medicaginisRoots-G. versiformeNodules-S. meliloti

ESTs1131731113

Overrepresentation Probability

ChiP = 0.03FisherP = 0.75ChiP = 3.4E-7FisherP = 0.46FisherP = 0.29FisherP = 0.44FisherP = 0.62FisherP = 0.26

Example: TC100493 Extension-like protein

TC100493 has an overrepresentation of ESTs from leaves infected with a virus or Colletotrichum

trifolli.

Criteria #4: Statistical analyses to identify soybean genes that are statistically over-represented among ESTs from pathogen or symbiont infected tissues in at least two EST libraries.

878 sequences identified

Criteria #5: Soybean genes that are statistically over-represented in at least one soybean EST library of pathogen infected tissues AND have homologs in other species that are also statistically over-represented in libraries of pathogen and/or symbiont infected tissues.

2,852 sequences identified

1,634 sequences from criteria 11,940 sequences from criteria 23,897 sequences from criteria 3878 sequences from criteria 42,852 sequences from criteria 5

Cross-referenced across all five experimental criteria to identify genes expressed in 2 or more experiments to give a final number of ~1,611 sequences.

Number of genes expressed in 2 experiments: 1,393Number of genes expressed in 3 experiments: 172Number of genes expressed in 4 experiments: 42Number of genes expressed in 5 experiments: 4

Putting it togetherIn total we found:

Narrowing the listUsing this list of all 1600 candidates,

Chunling chose around 200 candidates to start with based on Martijn’s ASR microarray data and keywords in the annotation

These 200 have been used to generate VIGS constructs

Gathering InfoBLASTN search against the phytoScaffold DB

The “Best” match is considered the targetThe other significant matches are considered the off

targetsBoth target and off targets may be silenced by the

VIGS construct, but we need to blast the VIGS sequence directly to be sure.

Used < e-4

BLASTX against the Uniref DBBest match is the gene we think our candidate isOther significant matches are in the same gene family

as our candidate gene, and may have similar functions.

Choosing New CandidatesUsing the current candidates and the master list of all

candidates, I wrote a short script that will update the master list by marking the genes that are in the candidate list

Using Steve’s criteria, I manually chose new candidate genes from the master list. The criteria consisted of keywords that matched in the candidates’ annotations. All genes in the same gene family as a match were also taken

BLASTN the new candidates against the phytoScaffold to determine target and off-targets for later use

Keywords used to pick new candidatesBURPF-boxkinaseMLOMYBphosphatasetranscription factorubiquitinZDS

Resulted in 207 new candidate genes

In ConclusionWet Lab

Learned wet lab techniques including topo cloning, transformation, and colony screening

Successfully screened 1 positive EST cloneDry Lab

Learned about ESTs, TCs, how to run BLAST searches and interpret the results

Collect BLAST information on the current VIGS candidates

Created a list of the next group of VIGS candidates

Thanks Steve, Chunling, and Michelle!