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Nature: The Team of Dr. Caixia Gao Creates new Disease-resistant and High-yielding Wheat Lines by Using Genome Editing Technology

Title: Genome-edited powdery mildew resistance in wheat without growth penalties

Journal: Nature

Impact factor (IF): 49.962

Vazyme collaboration products: AceQ® qPCR Probe Master Mix (Cat. No. Q112, for probe-based qPCR detection)

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Research Background

As one of the main food crops in the world, wheat feeds more than one-third of the world’s population. Powdery mildew is one of the major diseases affecting wheat production worldwide. Severely diseased wheat fields can reduce production by more than 40%, posing a serious threat to global food security.

As early as 2014, Dr. Caixia Gao’s team from the Institute of Genetics and Developmental Biology (IGDB) of the Chinese Academy of Sciences (CAS) and Dr. Jinlong Qiu’s team from the Institute of Microbiology cooperated to use genome editing technology to mutate the wheat susceptibility gene MLO, and obtain a wheat variety with broad-spectrum and durable resistance to powdery mildew1.

However, a new problem has arisen: due to the polymorphism of the gene, the MLO gene is not only a susceptibility gene for powdery mildew, but also affects other physiological characteristics of wheat. The researchers found that although wheat MLO mutants show resistance to powdery mildew, they also exhibit negative phenotypes such as premature senescence and reduced yield, which could limit their widespread use in production. The creation of wheat varieties that are both disease resistant and high yielding has therefore become a key challenge. Finally, after 8 years of collaborative research, the entire team has once again made important progress.

 

Paper overview

On February 9, 2022, the two teams collaborated again to publish a research paper in the Nature: Genome-edited powdery mildew resistance in wheat without growth penalties2.

This study elucidates the molecular mechanism of a novel MLO mutant in wheat with both disease resistance and high yield and uses genome editing technology to rapidly obtain an excellent new wheat line with broad-spectrum powdery mildew resistance and high yield. This work represents an important theoretical and technological breakthrough in the study of plant disease resistance breeding using the MLO susceptibility genes.

 

Research ideas and results

1. Found a mutant strain Tamlo-R32 with disease resistance and no growth defect

When screening the wheat mutant population of the MLO gene, the research team found a new MLO mutant, Tamlo-R32, which exhibited completely normal growth and yield while showing resistance to powdery mildew (Figure 1a-e). Whole-genome sequencing showed that the Tamlo-R32 mutant line had a large 304 kb deletion in the second exon of the TaMLO-B1 locus (Fig. 1f-g).

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Figure 1: Tamlo-R32 mutant lines exhibit disease resistance without affecting the growth and yield

2. The expression of the TaTMT3B gene was up-regulated in the Tamlo-R32 mutant strain

To study the impact of this large deletion, the research team used RNA-seq, qRT-PCR, and other methods to detect the expression of genes near the deletion site. The sequencing results revealed that the expression of the relevant genes was down-regulated or undetectable as expected. But it is worth noting that the expression of the TaTMT3B gene located upstream of the deletion site was significantly up-regulated (Fig. 2a-b). CUT&Tag and ATAC-seq were used to further detect histone modifications and chromosomal interactions at this locus. The results showed (Fig. 2c-e) that the Tamlo-R32 mutant had a large gene deletion near the TaMLO-B1 locus, altering the local chromatin state, thereby activating the up-regulated expression of TaTMT3B.

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Figure 2: Altered chromatin state in the Tamlo-R32 mutant resulted in up-regulated expression of the TaTMT3B gene

3. Verify the conserved function of TMT3 in Arabidopsis

To verify the function of the TaTMT3B gene, the research team knocked out TaTMT3B in the Tamlo-R32 mutant line. As a result, the plants recovered the undesirable phenotype of reduced height and reduced yield (Fig. 3a-c). Further overexpression of TaTMT3B in the yield-deficient mutant Tamlo-aabbdd. The results showed that the negative effects of plant height and yield disappeared, while powdery mildew resistance was not affected. Through gene knockdown and overexpression, it was verified that overexpression of TaTMT3B rescued the growth defect of the Tamlo-R32 mutant (Fig. 3d-h). Interestingly, the research team overexpressed AtTMT3 in the Arabidopsis Atmlo2/6/12 mutant, which also largely improve the leaf senescence phenotype in the mutant, indicating the functional conservation of the TMT3 gene (Fig. 3i).

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Figure 3: The expression of the TMT3 gene compensates for the phenotypic defect caused by the MLO mutant

4. Using genome editing technology to create excellent wheat varieties with disease resistance

After finding the mechanism by which the Tamlo-R32 mutation was able to maintain yield, the research team attempted to introduce the mutation into wheat. First, the Tamlo-R32 mutation was introduced into the main wheat cultivars by traditional breeding methods, and it was found that the powdery mildew resistance of these cultivars could be significantly improved. However, ordinary genetic transformation methods are time-consuming and labor-intensive. To accomplish this task more efficiently, the research team used CRISPR-Cas9 technology to achieve targeted mutation. Surprisingly, the research team succeeded in obtaining a wheat variety with broad-spectrum powdery mildew resistance in only 2-3 months without affecting growth and yield (Figure 4). This confirms the application value of the Tamlo-R32 mutant gene in production.

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Figure 4: Schematic of the rapid introduction of the Tamlo-R32 allele into high-quality wheat varieties by CRISPR-Cas9 technology

In conclusion, this study found a new Tamlo-R32 super allele in wheat that is resistant to disease without reducing yield. Through genome editing technology, an excellent new wheat variety with broad-spectrum powdery mildew resistance and high yield has been rapidly created. More importantly, the conserved function of the TMT3 gene in Arabidopsis was also confirmed, indicating that this idea can be applied not only to wheat but also has broad-spectrum potential in other crops. This provides a new strategy and technical route for breeding disease-resistant and high-yield crop varieties.

 

Vazyme’s Product Support

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Vazyme’s AceQ® qPCR Probe Master Mix (Cat. No. Q112) probe-based qPCR reagent facilitated this study. The researchers used the Q112 to verify and quantify chromatin interactions. This further revealed that the altered chromatin state in the Tamlo-R32 mutant would activate the molecular mechanism of up-regulated expression of the TaTMT3B gene.

AceQ® qPCR Probe Master Mix (Cat. No. Q112) is designed for high-specificity, high-sensitivity real-time quantitative PCR. The master mix contains AceTaq® HS DNA Polymerase, and with the optimized reaction system, it can effectively inhibit non-specific amplification, thereby significantly improving the amplification efficiency of PCR reaction, and enabling quantitative PCR to obtain a good linear relationship in a wide quantitative area. Compatible with various fluorescent probes, it is suitable for highly specific detection systems and can be used for multiplex fluorescence quantitative PCR detection. The package contains two different concentrations of ROX internal reference dye, which can be added according to the specific model used.

Vazyme is committed to serving customers with high-quality products, and relentlessly looking forward to working with more researchers to reach new heights in all areas of life sciences.

 

#Vazyme Q112 Product Information:

Product name and item number: AceQ® qPCR Probe Master Mix (Cat. No. Q112)

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Reference 

1. Wang Y , Cheng X , Shan Q , et al. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew[J]. Nature Biotechnology, 2014, 32(9):947-951.

2. Li S N, Lin D X, Zhang Y W, et al. Genome-edited powdery mildew resistance in wheat without growth penalties. Nature 602, 455–460 (2022). https://doi.org/10.1038/s41586-022-04395-9.