Experimental methods

Results

• PCR results show integration of the T-DNA fragment into 11 of the 16 mutants. • Target gene specific bands are present for six of the mutants; five of which are positive for T-DNA integration. • Presence of both T-DNA specific bands and gene specific bands will required future investigation into the genotypes of the fungal strains. • There was no significant reduction in foliar symptoms or root rot when mutants were compared with the Mont-1 transformation background. • There were symptoms found in the water treated plants which suggests contamination of water, seeds, or sand-soil, which may have lead to incorrect symptom readings.

References

Introduction Soybean sudden death syndrome (SDS) is a nationwide threat to soybean production and yield. In 2010, the fiscal detriment was valued at $0.82 billion nationally and $332 million in Iowa. SDS is caused by a fungal pathogen, Fusarium virguliforme. SDS produces both foliar and root symptoms. Foliar symptoms are characterized by interveinal chlorosis and necrosis; root symptoms include the darkening and rotting of the root tissues (Leandro et al., 2012). F. virguliforme is a pathogen that infects soybean roots. The pathogen does not localize in the foliar tissue, but systemic vascular transport of toxin is thought to cause foliar symptoms. One such toxin is FvTox1, a proteinaceous toxin produced by the pathogen (Brar et al., 2012). Fungal knockout mutants of the FvTox1 gene produce reduced foliar symptoms as compared with the wild type, Mont-1 (Pudake et al., 2013). While these knockouts show that FvTox1 is important for foliar symptoms, they did not show complete reduction in symptoms suggesting that there are other factors that play a role in foliar and root symptom development. Two approaches were used to determine other fungal proteins, which may play a role in SDS symptom development. Xylem sap from infected plants was collected and the protein content was compared to that of uninfected plants. There were five fungal proteins that were found in the infected plants (Abeysekara, Bhattacharyya, 2014). Targeted gene knockouts were created for these genes using T-DNA homologous recombination. The second approach looked at the genes that have increased expression during infection of soybean root when compared to their expression in germinating spores and mycelia. A list of candidate pathogenicity genes was developed from the highly expressed genes and homologous recombination mutants were created from some of these genes (Sahu et al., unpublished).

Foliar and root symptom assessment of soybeans inoculated with various Fusarium virguliforme mutants.

Kevin Schneider, Melissa Greene, Jordan Baumbach, Madan K. Bhattacharyya Department of Agronomy, Iowa State University, Ames, Iowa 50011

Fig. 2: Foliar disease symptom severity. Foliar disease symptoms were scored on a 1-7 scale as found below. Averages for three replications of 15 seedlings in each group are presented. Error bars show standard error between the three replications.

Fig. 3: Root disease symptom severity. Root disease severity was assessed by percentage of root showing root rot. Averages for three replications with 15 seedlings in each replication are presented. Error bars show standard error between three replications..

Objective •  Confirm the integration of T-DNA in 16 F. virguliforme targeted gene knockout mutants of 10

different F. virguliforme genes •  Screen 16 F. virguliforme knockout mutants for effects on pathogenicity

Acknowledgements This project was supported by the National Institute of Food and Agriculture, Grant no. 2013-68004-20374 by the United States Department of Agriculture (NIFA-USDA), and Iowa Soybean Association.

Harvest spores from 1/3 PDA plates

Grow fungal cultures in MSM media

PCR amplify fragments described in figure 2

Inoculate germinated soybean seeds with F. virguliforme Mont-1 or mutant spores

Plant inoculated seeds in sterilized sand:soil

Abeysekara NS, Bhattacharyya MK (2014) Analyses of the Xylem Sap Proteomes Identified Candidate Fusarium virguliforme Proteinacious Toxins. PLoS ONE 9(5): e93667. doi:10.1371/journal.pone.0093667

Brar H.K., Swaminathan S., and Bhattacharyya M.K. (2011) The Fusarium virguliforme toxin FvTox1 causes foliar sudden death syndrome-like symptoms in soybean. Mol. Plant Microbe Interact, 24: 1129-1188.

Leandro, L.F., N. Tatalovic, and A. Luckew. "Soybean Sudden Death Syndrome – Advances in Knowledge and Disease Management." CAB Reviews 7.053 (2012): 1-14. CAB Reviews. CABI. Web. 17 July 2015. <http://www.cabi.org/cabreviews/review/20123353118>.

Pudake, Ramesh N., Sivakumar Swaminathan, Binod B. Sahu, Leonor F. Leandro, and Madan K. Bhattacharyya. "Investigation of the Fusarium Virguliforme Fvtox1 Mutants Revealed That the FvTox1 Toxin Is Involved in Foliar Sudden Death Syndrome Development in Soybean." Current Genetics Curr Genet (2013): 107-17.

.

0

5

10

15

20

25

30

35

Aver

age

Roo

t Rot

Per

cent

0

1

2

3

4

5

6

Aver

age

Folia

r Sy

mpt

oms

hph Promoter Terminator F. virguliforme genome F. virguliforme genome

hph R

hph F Gene 5’

Gene 3’

Gene Function Knockout selection basis

FvTox Fungal toxin. Toxin responsible for foliar symptoms CW Cell wall protein with no known function. Fungal protein found in xylem sap MD Phytoalexin detoxification. Fungal gene upregulated during infection

SL Subtilase Fungal gene upregulated during infection

CP Cerato-platanin. Fungal protein found in xylem sap

LP Lipoprotein Fungal protein found in xylem sap

SS No known function. Fungal protein found in xylem sap PP No known function. Fungal protein found in xylem sap

AAP Amino Acid Permease Fungal gene upregulated during infection

PO Polyamine Oxidase. Fungal gene upregulated during infection

    PCR  Posi(ve  Knockout  Construct  

Promoter  Fragment  

Terminator  Fragment  

Gene  Specific  Fragment  

PO4-­‐3   +   -­‐   -­‐  PO2-­‐1   +   -­‐   -­‐  MD5-­‐5   -­‐   -­‐   -­‐  MD5-­‐3   +   -­‐   +  SL2   +   +   -­‐  SL20   -­‐   -­‐   -­‐  AAP5   +   +   +  Tox4   +   +   -­‐  Tox1   -­‐   -­‐   +  CP17   -­‐   +   -­‐  CP7   +   +   +  CW1   +   +   +  LP60   -­‐   +   -­‐  LP74   -­‐   -­‐   -­‐  PP34   -­‐   -­‐   -­‐  SS31   +   +   +  

Figure 1. Primer design for confirmation of the homologous in tegra t ion o f the T-DNA f r a g m e n t c o n t a i n i n g t h e hygromycin resistance gene hph. The gray arrows represent the F. virguliforme genome 5’ and 3’site of the intended T-DNA integration site. The purple boxes represent the genetic material integrated into the F. virguliforme genome including the promoter region of the targeted genes, the hph resistance gene and the terminator region of the targeted gene. Primers were designed for each knockout at the 5’end of the p r o m o t e r r e g i o n u s e d f o r homologous recombination, and 3’ end of the terminator region used for homologous recombination. Universal primers were also developed from the hph fragment of the T-DNA insert. The specific Gene 5’ primers were used in conjunction with the hph R primer to confirm the insertion of the promoter fragment. The specific Gene 3’ primers were used with the hph F primer to confirm the insertion of the terminator region in the knockouts. Additional primers specific to the targeted genes were used to check for the presence of the genes in the mutants.

Table 1. Functional categorization of knockout genes based on Blast analysis

Table 2. PCR results for the knockout mutants to confirm integration of T-DNA fragment.

+ Correct size bands present on gel - No bands present, or incorrect bands

Score foliar symptoms on a scale of 1-7

Score roots for percent root rot

Germinate sterilized Williams 82 seeds overnight in autoclaved water

Harvest mycelia and extract fungal DNA

Disease symptom screening T-DNA insertion confirmation

Foliar disease scoring: 1 = No symptoms 2 = Slight chlorosis 3 = Obvious intervienal chlorosis 4 = Leaf with necrosis on part of margin 5 = Necrosis along entire leaf margin 6 = Interveinal necrosis 7 = Complete leaf necrosis of leaf drop