Wheat Disease Resistance like Gene

A UNIQUE WHEAT DISEASE RESISTANCE LIKE GENE GOVERNS EFFETOR TRIGGERED SUCEPTIBILITY TO NECROTROPHIC PATHOGENS

ABSTRACT

Tan spot is an important foliar disease of wheat caused by Pyrenophora tritici-repentis. Eight races of this fungus have been identified based on their virulence on a wheat differential set. It is a devastating foliar disease of wheat caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis. Much has been learned during the past two decades about the genetics of wheat-P. tritici-repentis interactions. Research has shown that the fungus produces at least three host-selective toxins (HSTs), known as Ptr ToxA, Ptr ToxB, and Ptr ToxC, that interact directly or indirectly with the products of the dominant host genes Tsn1, Tsc2, and Tsc1, respectively. The primarily objective of this experiment was to study the susceptibility of the commercial cultivars to ToxA in in-vitro or inside the lab condition of the seedling stage.Among the four cultivars, Zippy, Magenta, LM-025 and Mace, Magenta is resistant to ToxA and zippy and LM-25 are susceptible varieties.The phylogenetic tree showed that two pathogens Pyrenophora tritici–repentis and P. nodorum are closely related to each other and are arrived from same ancestors. As, Magenta is resistant to the tanspot disease, it is highly suggested for the breeding of the variety.

Key Words: Pyrenophora tritici–repentis, P. nodorum; ToxA, Variety; Tanspot;

 INTRODUCTION

Wheat (Triticum aestivium L.) is a major winter season cereal crop grown at Australia. It is grown in all the states of the country occupying almost 20 million hectares of the productive land. In Western Australia alone, $2-3 billion wheat grain is generated among which 4200 family runs the wheat production ranging in size from 1000 to 15000 hectares.

There are two mains diseases that causes severe damages to the wheat crops which has got a symptom of necrotic and chlorotic disorder (yellowing of leaves) on the foliar parts. Pyrenophora tritici repentis and Parastagonospora nodorum are the casual agents behind these diseases. Yellowing of leaves are also termed as Tan spot. United states of America, Brasil, some parts of Africa and the south Asian countries that have high yield of wheat production are suffered by Pyrenophora tritici repentis.

ToxA is a 12 kDa protein that is coded with single copy gene ToxA. The ToxA is controlled by the gene namely Tsn1 that causes the death of the tissue of a host plant species (1). ToxB is 6·6 kDa protein that causes chlorosis in sensitive wheat genotypes and is encoded by the ToxB gene

 Disruption in photosynthesis, photosystem homeostatis are caused by these proteins showing effects. The presence of both Avirulence (Avr) gene and Plant resistance (R) gene causes the host resistance meaning it is incompatible to disease whereas absence of either one gene resulted in the susceptibility which is compatible of host to disease.

Pyrenophora tritici-repentis (Ptr), causal agent of tan spot of wheat, is a necrotrophic fungus that presents an increasing threat to wheat production due to its rapid, global expansion. Despite its homothallic nature, Ptr populations have high genetic diversity, which positively impacts host range and virulence. In Queensland, about 50% crop loss was susceptible cultivars of wheat. (Tsn1, Tsn2) With the presence Tsn1, Tsn2, virulence is observed in the wheat whereas to aid the breeding in the resistance to tan spot, the absence of sensitive loci was observed.

This experiment was overall done in close consideration to inspect the selective wheat variety were or were not capable of ToxA. Additionally, it was also done in order to know if there were any horizontal transfer of ToxA to the Tsn1 in between the two-causal agent; Pyrenophora tritici repentis and Parastagonospora nodorum

MATERIALS AND METHODS

Wheat Cultivars

The study was carried out using Qiagen DNeasy Plant Mini Kit for the process of DNA extraction and the entire observation was done using four commercial varieties that included Mace, Magenta HLM 025 and a sensitive variety namely Zippy. A ready to go seedings were provided during the lab week at Curtin University.

Wheat Leaf Infiltration

All four Mace, Magenta HLM 025 and a sensitive variety namely Zippy were used for the purpose of leaf infiltration. Zippy is Tsn1 positive whereas Mace is negative control and other two are unknown cultivars. Firstly, the leaves were selected, sliced into small pieces and then drenched into the vile. It was soaked for around 2 to 3 centimetres from the top of the leaves surface of the leaf. Then the leaves were marked. Similarly, 50 µg/ml of ToxA was infiltrated into the first leaf and soaked area was marked again with a different markingwith two replicates for each of the varieties. In total, 16 infiltrated plants were obtained, eight with water and eight with ToxA and labelling was done with group number and that was 14.

DNA Extraction

All the three samples (zippy, magenta and LM 025) were chopped in a very small pieces and was kept inside 2ml of microcentrifuge tube. They were then labelled using varieties, group number and most importantly the replications that were R1,2,3. It was kept for 5 minutes and then 400ul of buffer solution and 4ul of RNase was added. Using a micro pestle, it was grinded until paste or water substance was seen. Then, it was vortexed for 2 minutes and incubated at 65-degree centigrade inverting it thrice for cell lysis during this process. Again, 130ul of buffer was then added in the same mixture which was then incubated inside the ice for five minutes. It was done so that it would precipitate the detergent, proteins and also the polysaccharide. The Lysate was centrifuged at 14000RPM for five minutes and then was pipetted into the QIA shredder mini spin coredder mini spin column. The buffer solution AW1 in lysate was added @ 1.5 volume and was mixed by the use of pipette. 650ul of the mixture was pipetted and was again centrifuged at 800 RPM for one minute.  The solution collected was thrown and same steps was repeated for remaining mixture. That DNeasy mini spin column was placed into new 2ml collection tube with the addition of 500ul buffer and was placed for centrifugation @ 8000 RPM for one minutes. The solution was thrown, and collection tube was reused for the addition of 500ul of buffer to mini spin column which was centrifuged @ 14000RPM for two minutes to dry the membrane.  After the transfer, mini spin column into microcentrifuge tube, 100ul of buffer was pipetted into the membrane along with the incubation for five minutes at room temperature and centrifuged at 8000 RPM for an another minute.

Polymerase Chain Reaction (PCR) of wheat DNA using Tsn1 primers

For Polymerase Chain Reaction (PCR), DNA was extracted from the six wheat leaves of three cultivars (Zippy, Magenta and LM-025) using Qiagen DNeasy plant mini-kit following the manufactures guidelines. The extracted DNA was used to test for the presence of Tsn1 gene in the cultivars using molecular marker.

A ready-made master mix was set up for PCR in such a way that mixture would be sufficient for running 10 PCR reactions. Total 160ul of master mix (50ul of Cresol red, 480ul of MilliQ water, 20ul of primers, 2ul of Taq DNA polymerase and 40ul of Taq buffer) was prepared and was kept on ice. After that DNA was pipetted into 6 separate 0.2ml PCR tubes. 4ul of Each sample was used for PCR with the addition of 16ul of master mix in each DNA samples and PCR was run in PCR machine.

Agarose Gel Electrophoresis of PCR Amplified Products

Agarose gel electrophoresis is another way to quickly estimate DNA concentration as it visualizes DNA fragments in different sizes. In order for the electrophoresis to run,the gel tank was filled with TAE buffer and was comb was gently removed from the tank. The PCR product (DNAs) were taken and was loaded into the agarose gel each 10ml that was recorded. Later, after a while, 2.5ul of ladder was poured into last well of agarose gel and was kept for electrophoresis at voltage of 80. At the point when all the DNA had migrated towards the well, voltage was turned up to 110 until DNAs had migrated downwards to the two third of the way through the gel.

 Alignment of Tsn1 sequence, PtrToxA sequences and BLAST analysis

A thorough computer-based programming software “Geneious” was used for these processes. Nucleotide alignment and sequences of Tsn1 of wheat varieties was formed. For the PtrToxA sequences, the gene bank database was used where PtrToxA mRNA sequence was translated into protein sequence and BLAST analysis was carried out from NCBI homepage.

Fungal phylogeny construction

Phylogenetic tree using ribosomal gene was formed for the determination of the relationship between the fungi that contain the PtrToxA sequence. With the use of Geneious software and LSU sequence from 20 fungi including P.tritici repentis and P. nodorum. The sequences obtained from this were then aligned in the tree structure along with their genetic distances. This took a while to be noted and understood but was however accomplished at the end.

RESULTS

Quantification of DNA using nanodrop:

In all cultivars of wheat, the ratio of 260/280 was found to be higher than 2.0 that showed the presence of RNA in the samples except in Zippy where the DNA was present as ratio was 1.78. The ratio 260/230 gave lower values than 2.0-2.2 that resulted the contamination of DNA with organic compounds and also human contamination like saliva.

DNA Sequencing:

Use of the gel electrophoresis for DNA sequencing was found to be negative.

Susceptibility of Wheat varieties with the infiltration to ToxA

Wheat variety Zippy was found to be susceptible to ToxA where necrotic spots were seen in infiltrated leaves with the ToxA. But Magenta variety was resistant to ToxA as no effect was seen in infiltrated leaves. In case of varieties LM-025 and Mace, Mace is more susceptible to ToxA than LM-025 as more necrotic spots was seen in LM-025 than in Mace. Scout was half insensitive to ToxA.

Construction of fungal phylogeny

The fungal species Pyrenospora tritici-repentis and Paragonospora nodorum have been found to be closely related species by phylogenetic tree.

 Horizontal transfer of gene between two fungi was determined. The genetic distance between these two species was 0.201.  Identity and evaluation to PTR is 100% and 0.0 respectively and 99% and 0.0 for P. nodorum.  

 BLAST analysis

Pyrenophora tritici-repentis and Paragonostophora nodorum are seen to be closely related species. The value of both species was 0.0 and similarity for Ptr was 100% and 99% for P. nodorum.

Tsn1 Alignment:

We were unable to find out the Tsn1 alignment because of the failure of PCR but we were told that the reason was a technical error of the PCR functioning during the lab session.

DISCUSSION

The DNA quantification using nanodrop showed higher ratio of 260/280 that should be around 1.8 for the pure DNA. There was presence of RNA in higher amount than DNA as the ratio was higher than 2 in all cases. For the 260/230 test, the value from 2 to 2.2 is suitable for getting of pure DNA. In all cultivars, the value was found to be lower which denoted that there was a massive contamination of sample. There may be presence of more organic compounds than DNA. DNA sequencing was found to be negative that may be due to the improper running of PCR and also the contamination of samples by RNA and organic compounds.

The infiltration of ToxA in four cultivars of wheat (Magenta, Zippy, Mace and LM-025) provided the result that Magenta was insensitive/ resistance to ToxA whereas Zippy was susceptible to ToxA. There was presence of necrotic spots in the leaves in areas where ToxA was infiltrated. Magenta cultivar can be used for the further breeding purpose and also can be recommended to farmers for its cultivation. Cultivar Mace was susceptible to ToxA and Scout was found to be half insensitive to ToxA. Interaction between ToxA and Tsn1 gene can only develop the necrotic spot. Absence of Tsn1 gene in wheat cultivar will not be able to form tan spots with the infiltration of ToxA. Various studies also provided the findings that wheat cultivars contained Tsn1 gene.

Wheat gene Tsn1 recognizes the host specific toxin ToxA, where presence of both HST and Tsn1 gene develops the disease in wheat plant. Wheat cultivars of Australia has shown high proportion of Tsn1 gene due to which wheat cultivars are susceptible to tan spot disease. Thus, for the resistance, the wheat breeder should counter select tsn1 for the improvement of resistance in wheat cultivars⁸. Breeding purposes using resistant gene of cultivars and use of wild species of wheat cultivars (T. monococum, T. dicoccum) can help for the resistance to tan spot disease⁷.

The phylogenetic tree showed that two pathogens Ptr and P. nodorum are closely related with eachother arising from same anscestors. There can be horizontal transfer of Tsn1 gene between species.

CONCLUSION

Overall, the wheat variety Zippy, Yitpi and Scout are susceptible to ToxA due to the presence of gene Tsn1 and only Magenta variety is resistant to ToxA.  Fungal species Pyrenophora tritici-repentis and Paragonospora nodorum are closely related species that was found in phylogenetic tree. There can be horizontal transfer of Tsn1 gene between the two species as evalue was found to be 0.0 and percentage similarity was 100 in Ptr and 99% in P. nodorum.

RECOMMENDATIONS

Wheat variety Magenta can be used for breeding purposes and can be recommended to farmers for growing as it is resistant to ToxA. Further research activities can be done for necrotic spots and Tsn1 gene as Tsn1 gene has horizontal transfer between the species that can flourish the disease which should be controlled for the better production of wheat with the resistance to disease.

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FIGURES

Figure No. 1: Susceptibility of wheat cultivars (Magenta, Zippy, LM-025 and Mace) to ToxA with the infiltration of ToxA in wheat leaves.

Figure No. 2:  Tsn1 Alignment

Figure No. 3: Amplification of Tsn1 in Agarose gel

 Figure No. 1

Figure No. 2

Fig. 3: Amplification of Tsn1 gene in Agarose gel