Clavibacter michiganensis subsp. Nebraskensis (Cmn) is a pathogen responsible for Goss’s Wilt in maize in the high plains. Strains of Clavibacter michiganensis subsp. Nebraskensis that are virulent and non-virulent in maize have been isolated, but the mechanism of this differentiation is not understood. Investigation of the genetic differences between virulent and non-virulent strains is providing an explanation as to how Cmn causes disease. Cmn mutants with reduced or removed virulence have been created using Tn5 transposon transformation to randomly knockout virulence factors. Potential effectors have also been identified in a genomic “virulence island” region by using bioinformatics

Alfano, James R.

Eastman, Samuel

Herr, Josh

Li, Guangyong

Yang, Fan

English

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University of Nebraska - Lincoln
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Molecular Mechanisms of Goss’s Wilt
Samuel Eastman
University of Nebraska-Lincoln, eastman_samuel@yahoo.com
Guangyong Li
University of Nebraska-Lincoln, gli3@unl.edu
Fan Yang
University of Nebraska-Lincoln, fyang@huskers.unl.edu
Josh Herr
University of Nebraska-Lincoln, jherr@unl.edu
James R. Alfano
University of Nebraska-Lincoln, jalfano2@unl.edu
Follow this and additional works at: http://digitalcommons.unl.edu/ucareresearch
Part of the Plant Pathology Commons
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Eastman, Samuel; Li, Guangyong; Yang, Fan; Herr, Josh; and Alfano, James R., "Molecular Mechanisms of Goss’s Wilt" (2016).
UCARE Research Products. 30.
http://digitalcommons.unl.edu/ucareresearch/30
Molecular Mechanisms of Goss’s Wilt
Samuel Eastman1, Guangyong Li2,3, Fan Yang2,3, Josh Herr2,3 and James R. Alfano1,2,3
1UNL Undergraduate Microbiology,
2Center for Plant Science Innovation and 3Department of Plant Pathology, University of Nebraska-Lincoln
Abstract
Clavibacter michiganensis subsp. Nebraskensis (Cmn) is a 
pathogen responsible for Goss’s Wilt in maize in the high plains.
Strains of Clavibacter michiganensis subsp. Nebraskensis that are 
virulent and non-virulent in maize have been isolated, but the 
mechanism of this differentiation is not understood. Investigation of 
the genetic differences between virulent and non-virulent strains is 
providing an explanation as to how Cmn causes disease. Cmn
mutants with reduced or removed virulence have been created using 
Tn5 transposon transformation to randomly knockout virulence 
factors. Potential effectors have also been identified in a genomic 
“virulence island” region by using bioinformatics. 
Gene Knockout using Homologous Recombination
Using Homologous Recombination the virulence region described 
above and all the genes contained within will be knocked out all at 
once. A specially-prepared version of DNA with identical sequences 
from up- and down-stream of the virulence region will bind to the 
genomic DNA and replace the region with a selectable antibiotic 
marker
Reverse Genetics Approach
By comparing the sequence of the virulent strain of Cmn to other, 
avirulent strains, candidate genes for potential effectors were 
identified. The genes in green are located clustered together with a 
gene for a Phage integrase protein, suggesting a region of virulence 
genes (a “virulence island”), responsible for the acquisition of 
pathogenicity and Goss’s Wilt symptoms.
Acknowledgements
We appreciate the help of Dr. Bob Harveson and Kathy Nielson for 
providing heirloom Golden Bantam maize seeds and Cmn strains from 
their collection, of Dr. Tamra Jackson for providing many base strains of 
Cmn from her collection, and of Dr. Riekhof for assistance with 
molecular techniques.
This research was supported by a UCARE grant from the University of 
Nebraska-Lincoln.
Strain-Specific Colony PCR
By including strain-specific primers in the PCR reaction, potentially avirulent 
strains were either identified as arising from the original transformed strain, 
or identified as a contaminant. 
Clavibacter Virulence Data
Virulence readings on over 1000 randomly generated mutants were taken 
and recorded. Potentially avirulent strains are shown in green to show 
reduced or absent virulence in the plant.
Goss’ Wilt Symptoms
Goss’s Wilt is a vascular pathogen that causes tissue necrosis parallel to 
leaf veins, with characteristic spots of bacterial exudate. Yield loss due to 
Goss’s Wilt can reach 50% in severely infected fields. Goss’s Wilt is 
historically a Nebraska pathogen but the range of disease increases 
during hot, dry years.
Inoculated corn symptom comparison
The severity of Goss’s Wilt symptoms was recorded in all inoculated 
plants. Transformed strains that caused significantly reduced or absent 
symptoms were flagged for analysis and further testing.
Laboratory Setup
Plants were inoculated by dabbing bacterial solution on a leaf cut. They 
were then kept for 1 week in a growth chamber at 24 degrees C.
Pro. ID Gene Name Character Size(bp) GC% GC Diff.(-73%)
PROKKA_00090 hypothetical protein unknown 93 73.1 0.1%
PROKKA_00133 hypothetical protein 210 79 6.0%
PROKKA_00432 Phage_integrase Phage_integrase 1116 63.9 -9.1%
PROKKA_00434 hypothetical protein unknown 372 65.05 -8.0%
PROKKA_00435 hypothetical protein unknown 243 59.25 -13.8%
PROKKA_00441 hypothetical protein unknown 408 65.2 -7.8%
PROKKA_00442 hypothetical protein unknown 420 65.48 -7.5%
PROKKA_01195 hypothetical protein unknown 138 73.2 0.2%
PROKKA_01305 hypothetical protein unknown 990 51 -22.0%
PROKKA_01416
BsuMI modification methylase subunit 
YdiO
putative BsuMI modification methylase 
subunit YdiO 1140 57.63 -15.4%
PROKKA_01495 hypothetical protein unknown 120 79.2 6.2%
PROKKA_01504 hypothetical protein unknown 183 75.41 2.4%
PROKKA_01554 hypothetical protein unknown 156 78.2 5.2%
PROKKA_02089 hypothetical protein unknown 246 73.2 0.2%
PROKKA_02139 hypothetical protein unknown 117 75.21 2.2%
PROKKA_02246 hypothetical protein unknown 312 81.93 8.9%
PROKKA_02310 hypothetical protein unknown 135 77.8 4.8%
PROKKA_02646 hypothetical protein unknown 294 65 -8.0%
PROKKA_02648 hypothetical protein unknown 174 67.8 -5.2%
PROKKA_02652 hypothetical protein unknown 246 65.9 -7.1%
PROKKA_02653 hypothetical protein unknown 402 68.7 -4.3%
PROKKA_02654 hypothetical protein unknown 513 67.3 -5.7%
PROKKA_02655 hypothetical protein unknown 180 65 -8.0%
PROKKA_02681 hypothetical protein unknown 99 57.6 -15.4%
Strain Virulence Date Inoculated Mutant Type
Method of 
Inoculation
Type of Corn Location
511 + 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
512 + 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
513 + 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
514 + 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
515 + 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
516 + 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
517 + 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
518 + 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
519 - 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
Tn5 Transposome Random Mutagenesis
By transforming Cmn with a transposon carrying a selectable antibiotic-
resistance gene, over a thousand random mutants of Cmn were 
created. The Tn5 transposon inserts randomly into the genome, 
breaking any gene in which it lands.
Ctrl 451 519 566 573 575 576
Strain Virulence Date Inoculated Mutant Type
Method of 
Inoculation
Type of Corn Location
519 - 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
562 - 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
573 - 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
575 - 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
576 - 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
586 - 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
623 - 2/19/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
628 - 2/19/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
658 - 2/19/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
681 - 2/19/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
720 - 2/26/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
721 - 2/26/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC
Avirulent Mutants
Out of almost 1200 Tn5 mutants, 12 showed no virulences on maize


Molecular Mechanisms of Goss’s Wilt

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University of Nebraska - LincolnDigitalCommons@University of Nebraska - LincolnUCARE Research Products UCARE: Undergraduate Creative Activities &Research Experiences4-2016Molecular Mechanisms of Goss’s WiltSamuel EastmanUniversity of Nebraska-Lincoln, eastman_samuel@yahoo.comGuangyong LiUniversity of Nebraska-Lincoln, gli3@unl.eduFan YangUniversity of Nebraska-Lincoln, fyang@huskers.unl.eduJosh HerrUniversity of Nebraska-Lincoln, jherr@unl.eduJames R. AlfanoUniversity of Nebraska-Lincoln, jalfano2@unl.eduFollow this and additional works at: http://digitalcommons.unl.edu/ucareresearchPart of the Plant Pathology CommonsThis Poster is brought to you for free and open access by the UCARE: Undergraduate Creative Activities & Research Experiences atDigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in UCARE Research Products by an authorized administratorof DigitalCommons@University of Nebraska - Lincoln.Eastman, Samuel; Li, Guangyong; Yang, Fan; Herr, Josh; and Alfano, James R., "Molecular Mechanisms of Goss’s Wilt" (2016).UCARE Research Products. 30.http://digitalcommons.unl.edu/ucareresearch/30Molecular Mechanisms of Goss’s WiltSamuel Eastman1, Guangyong Li2,3, Fan Yang2,3, Josh Herr2,3 and James R. Alfano1,2,31UNL Undergraduate Microbiology,2Center for Plant Science Innovation and 3Department of Plant Pathology, University of Nebraska-LincolnAbstractClavibacter michiganensis subsp. Nebraskensis (Cmn) is a pathogen responsible for Goss’s Wilt in maize in the high plains.Strains of Clavibacter michiganensis subsp. Nebraskensis that are virulent and non-virulent in maize have been isolated, but the mechanism of this differentiation is not understood. Investigation of the genetic differences between virulent and non-virulent strains is providing an explanation as to how Cmn causes disease. Cmnmutants with reduced or removed virulence have been created using Tn5 transposon transformation to randomly knockout virulence factors. Potential effectors have also been identified in a genomic “virulence island” region by using bioinformatics. Gene Knockout using Homologous RecombinationUsing Homologous Recombination the virulence region described above and all the genes contained within will be knocked out all at once. A specially-prepared version of DNA with identical sequences from up- and down-stream of the virulence region will bind to the genomic DNA and replace the region with a selectable antibiotic markerReverse Genetics ApproachBy comparing the sequence of the virulent strain of Cmn to other, avirulent strains, candidate genes for potential effectors were identified. The genes in green are located clustered together with a gene for a Phage integrase protein, suggesting a region of virulence genes (a “virulence island”), responsible for the acquisition of pathogenicity and Goss’s Wilt symptoms.AcknowledgementsWe appreciate the help of Dr. Bob Harveson and Kathy Nielson for providing heirloom Golden Bantam maize seeds and Cmn strains from their collection, of Dr. Tamra Jackson for providing many base strains of Cmn from her collection, and of Dr. Riekhof for assistance with molecular techniques.This research was supported by a UCARE grant from the University of Nebraska-Lincoln.Strain-Specific Colony PCRBy including strain-specific primers in the PCR reaction, potentially avirulent strains were either identified as arising from the original transformed strain, or identified as a contaminant. Clavibacter Virulence DataVirulence readings on over 1000 randomly generated mutants were taken and recorded. Potentially avirulent strains are shown in green to show reduced or absent virulence in the plant.Goss’ Wilt SymptomsGoss’s Wilt is a vascular pathogen that causes tissue necrosis parallel to leaf veins, with characteristic spots of bacterial exudate. Yield loss due to Goss’s Wilt can reach 50% in severely infected fields. Goss’s Wilt is historically a Nebraska pathogen but the range of disease increases during hot, dry years.Inoculated corn symptom comparisonThe severity of Goss’s Wilt symptoms was recorded in all inoculated plants. Transformed strains that caused significantly reduced or absent symptoms were flagged for analysis and further testing.Laboratory SetupPlants were inoculated by dabbing bacterial solution on a leaf cut. They were then kept for 1 week in a growth chamber at 24 degrees C.Pro. ID Gene Name Character Size(bp) GC% GC Diff.(-73%)PROKKA_00090 hypothetical protein unknown 93 73.1 0.1%PROKKA_00133 hypothetical protein 210 79 6.0%PROKKA_00432 Phage_integrase Phage_integrase 1116 63.9 -9.1%PROKKA_00434 hypothetical protein unknown 372 65.05 -8.0%PROKKA_00435 hypothetical protein unknown 243 59.25 -13.8%PROKKA_00441 hypothetical protein unknown 408 65.2 -7.8%PROKKA_00442 hypothetical protein unknown 420 65.48 -7.5%PROKKA_01195 hypothetical protein unknown 138 73.2 0.2%PROKKA_01305 hypothetical protein unknown 990 51 -22.0%PROKKA_01416BsuMI modification methylase subunit YdiOputative BsuMI modification methylase subunit YdiO 1140 57.63 -15.4%PROKKA_01495 hypothetical protein unknown 120 79.2 6.2%PROKKA_01504 hypothetical protein unknown 183 75.41 2.4%PROKKA_01554 hypothetical protein unknown 156 78.2 5.2%PROKKA_02089 hypothetical protein unknown 246 73.2 0.2%PROKKA_02139 hypothetical protein unknown 117 75.21 2.2%PROKKA_02246 hypothetical protein unknown 312 81.93 8.9%PROKKA_02310 hypothetical protein unknown 135 77.8 4.8%PROKKA_02646 hypothetical protein unknown 294 65 -8.0%PROKKA_02648 hypothetical protein unknown 174 67.8 -5.2%PROKKA_02652 hypothetical protein unknown 246 65.9 -7.1%PROKKA_02653 hypothetical protein unknown 402 68.7 -4.3%PROKKA_02654 hypothetical protein unknown 513 67.3 -5.7%PROKKA_02655 hypothetical protein unknown 180 65 -8.0%PROKKA_02681 hypothetical protein unknown 99 57.6 -15.4%Strain Virulence Date Inoculated Mutant TypeMethod of InoculationType of Corn Location511 + 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC512 + 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC513 + 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC514 + 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC515 + 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC516 + 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC517 + 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC518 + 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC519 - 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GCTn5 Transposome Random MutagenesisBy transforming Cmn with a transposon carrying a selectable antibiotic-resistance gene, over a thousand random mutants of Cmn were created. The Tn5 transposon inserts randomly into the genome, breaking any gene in which it lands.Ctrl 451 519 566 573 575 576Strain Virulence Date Inoculated Mutant TypeMethod of InoculationType of Corn Location519 - 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC562 - 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC573 - 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC575 - 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC576 - 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC586 - 2/12/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC623 - 2/19/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC628 - 2/19/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC658 - 2/19/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC681 - 2/19/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC720 - 2/26/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GC721 - 2/26/16 EzTn5 Mutant (2) Nick-Dab Heirloom GB GCAvirulent MutantsOut of almost 1200 Tn5 mutants, 12 showed no virulences on maize

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Molecular Mechanisms of Goss’s Wilt

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