After eight years, the "anti-murder case" of wheat powdery mildew, which has attracted widespread attention, has finally ushered in a sequel.
Gao Caixia's team from the Institute of Genetic Development of the Chinese Academy of Sciences and Qiu Jinlong's team from the Institute of Microbiology of the Chinese Academy of Sciences used multiple "gene scissors" to achieve accurate control of the important disease-sensitive gene sequences of wheat, and obtained new materials with high resistance to powdery mildew and high yield. The study was published in Nature in the early hours of February 10. It means that powdery mildew, which is known as one of the three major diseases of wheat, has finally been "taken down" by mainland scientists.
"This research work, which has important theoretical and practical application value, will become a landmark achievement in the field of crop breeding." Academician Kang Zhensheng of Northwest A&F University commented that it shows the great potential of genome editing in crop molecular design and breeding, which is of great significance to ensuring food security.
"The other boot" landed on the ground
Powdery mildew is one of the three major diseases that widely affect wheat yields. According to statistics from the Ministry of Agriculture and Rural Affairs, the area of wheat affected by powdery mildew on the mainland reaches about 100 million mu every year, and the yield of seriously ill fields will even be reduced by 40%. "Bringing to justice" this fungus, which poses a serious threat to food security, is the dream of many breeding experts.
The trend chart of the three major diseases of wheat can be seen in the figure that the incidence area of wheat powdery mildew remains high. Courtesy of the author
Currently, molecular breeders are resistant to powdery mildew through resistance genes. But like virus prevention, this pathway is not broad-spectrum and persistent, and it is easy to lose its effectiveness with the emergence of new subspecies of powdery mildew.
Successful infection of pathogenic bacteria requires the use of plant susceptibility genes, can a broad spectrum of long-lasting resistance be obtained by blocking the "bridge" between this disease and the plant? For a long time, this was an unattainable dream for the scientific community.
"Mutations in the disease-susceptible gene are often able to give plants broad-spectrum and long-lasting disease resistance, but it often has important physiological functions, and its mutations can bring a variety of negative effects to plant growth and development." Li Shengnan, the first author of the paper and an assistant researcher in Qiu Jinlong's team, explained to reporters that this further reflects the "cunning" of pathogenic bacteria and greatly limits the application of disease sensitivity genes in plant disease resistance breeding.
Photo of Li Shengnan. Courtesy of respondents
However, Gao Caixia's team and Qiu Jinlong's team combined their advantages in gene editing and disease resistance research, and chose to use new theories and advanced biotechnology of plant disease resistance to overcome the problems facing breeders and plant biologists.
Scientists have long known that MLO is a disease-sensitive gene in wheat, but because ordinary wheat is heterologous hexaploid, the MLO gene has 3 copies, and it is almost impossible to knock out these three genes at the same time by natural mutations. In 2014, the collaborative team used "genetic scissors" to knock out three copies of MLO in a targeted manner, unsurprisingly obtaining a new wheat material with broad-spectrum persistent resistance to powdery mildew.
The research received a great deal of attention worldwide after the publication of Nature Biotechnology. The study was selected as one of the 20 most influential articles in the journal's 20th anniversary and was hailed by MIT Technology Review as "one of the world's top ten technological breakthroughs." Gao Caixia was also selected as one of the "Ten Chinese Science Stars" in Nature's 2016 year for leading the wave of plant genome editing.
However, there is another half of the story — as observed in many other plants, and the team also found that wheat knocked out the disease-susceptible gene MLO showed a certain degree of negative phenotype, such as premature aging, plant shorter, yield decline, etc., limiting its widespread use in production. However, the research team chose to face the difficulties.
Lucky always favored those who worked hard, and among the more than 100 genome-edited wheat mutants obtained after knocking out MLO at that time, they found a "baby" material, the mutant Tamlo-R32. While it exhibits resistance to powdery mildew, growth, development and yield are completely normal.
This distinctive material makes Gao Caixia firmly believe that the disease-inducing gene mutation resistance is not a "dead end" and that "walking along this road will definitely be done".
Now, after eight years of joint efforts, "another boot" has finally landed. In a new study published in Nature, they unraveled the secret behind the Tamlo-R32 mutation, overcame the negative phenotype caused by the MLO mutation in the disease-susceptible gene, and achieved a combination of disease-resistant and high-yield "fish and bear paws" at the same time.
Layer by layer to advance to break the suspense
Why does Tamlo-R32 stand out among the large number of mutants obtained by knocking out MLO? How did this material, which yields even more than ordinary wild-type wheat, come about? How can it be achieved through genome editing and introduced into the main wheat cultivar?
After 2014, the series of suspense around tamlo-R32, the "protagonist", became a puzzle that Gao Caixia and her collaborators urgently wanted to solve.
It's not easy.
The ordinary wheat genome is very large, 5 times the human genome and 40 times the rice genome. Its sequence repeatability is quite high, and the genome structure is extremely complex, which poses great challenges to unravel the mysteries that cause Tamlo-R32 mutants.
Initially, because the wheat genome data was not perfect, the research team could only analyze it through a long series of traditional genetic experiments, which allowed them to determine that there were expected mutations in the A and D genomes of wheat's three chromosome groups A, B, and D.
"But if only these two genomes are changed, it is not enough to fight powdery mildew, so there must be problems on the B genome." Gao Caixia said that due to the genomic data at the time, the research team explored this problem for four years and never got a glimpse of its key.
It wasn't until 2018, with the help of newly completed wheat genome resequencing data and chromosomal fine maps, that this "dark box" was finally opened.
To the surprise of Gao Caixia and her collaborators, a large deletion of up to 304 Kb (more than 300,000 DNA letters) occurred on the B genome of the Tamlo-R32 mutant – which led to the three-dimensional structure of the mutant's chromosomes being altered, which increased the expression level of the upstream gene TaTMT3 (related to the sugar transporter), thus overcoming the negative phenotype caused by the MLO mutation of the disease-susceptible gene, and finally achieving a win-win situation of disease resistance and yield.
Comparison of wild-type, Tamlo-R32 mutant, and knockout MLO susceptibility gene wheat. The left panel is wild-type, where TaTMT3 expression is suppressed; the right panel is a Tamlo-R32 mutant, and the expression of TaTMT3 is activated after deleting a large fragment of the genome of 304Kb. Courtesy of the author
The suspense is solved, but it is not easy to accurately cut the large genomic fragment of 304Kb. "This requires the 'scissors' to be particularly efficient." Gao Caixia told reporters that in the past ten years of anti-powdery mildew gene editing research, it has now possessed 7 core technology patents, and the research team has also created a series of new genome editing technologies.
It is based on these core technologies that the research team used "gene scissors" by superposition to delete large fragments of DNA near TaMLO-B while knocking out the MLO disease-inducing gene, thus realizing the introduction of this disease resistance and high yield excellent trait into many wheat main varieties in mainland China.
Since the gene function of MLO is relatively conserved in different plants. The researchers further found that overexpression of TMT3 in the model plant Arabidopsis thaliana also overcame the negative phenotype produced by mutations in its susceptible gene. "This proves that superimposed genetic changes can overcome the growth defects caused by disease-inducing gene mutations, and provides a new theoretical perspective for crop disease resistance breeding research." Li Shengnan said.
At this point, the research team finally finished telling the story of using disease-susceptibility genes for wheat disease resistance breeding. Looking back on the challenges, Gao Caixia said with some lightness: "We know that the road is there, as long as we persevere, we will definitely be able to reach it." ”
She particularly emphasized Li Shengnan's efforts in this process: "He is really working hard. Other people in the same lab have published a lot of articles and become professors. He went from postdoctoral fellow to assistant researcher and has been doing this consistently. ”
Kung Fu pays off. This research result, which has been achieved after the "Eight-Year War of Resistance", has been unanimously praised by the reviewers. Multiple reviewers described the study as "exciting" and "has a lot of potential for application." One of the reviewers noted: "This work represents an important step forward in the exploration of disease-resistant wheat without negative effects. ”
Gao Caixia photo. Courtesy of respondents
Leading the next generation of breeding
With nearly eight years of "dead end" basic research, Gao Caixia and her collaborators have a clear purpose: to move towards the application of genome editing, so that powdery mildew is no longer a threat to wheat.
"One of the advantages of genome editing is that crop breeding and improvement can be carried out more easily, quickly and accurately." Li Shengnan said that the research team spent several years to understand the mutation mechanism of Tamlo-R32, but it took only a few months to use genome editing technology to obtain disease-resistant and high-yield germplasm resources in multiple wheat main cultivars. Traditional hybrid breeding takes five or six years.
In field trials conducted in Beijing and Zhao County, Hebei Province in 2019 and 2020, the joint team further demonstrated the reliability of new germplasm resources: conventional MLO mutants caused plant height dwarfing by about 10% and yield reductions of about 16%, while new mutants had yields that exceeded or at least maintained consistency with parents.
"Cultivating and promoting new disease-resistant varieties is the most economical, efficient and environmentally friendly strategy for controlling plant diseases." Academician Kang Zhensheng commented, "This study verifies that the development of genome editing technology has a significant role in promoting the improvement of crop traits, especially the improvement of polyploid complex genome crops that are difficult to implement classical genetic modification, which is of great significance to ensuring food security." ”
"Compared with traditional breeding techniques, the advantages of genome editing breeding are very obvious." Gao Caixia said that the introduction of a disease resistance gene in traditional hybrid breeding requires 6 to 8 generations of backcrossing, the whole process is very long, and the premise is that the hybrid parent species must have disease resistance genes. Through mutation breeding (radiation, chemical mutagenesis, etc.) is blind and random, finding the ideal mutant is tantamount to finding a needle in a haystack. Genome editing offers the possibility of precisely targeted breeding.
"Through genome editing, you can not add any exogenous genes, only need to modify the targeted sequence, which greatly saves time and workload." She added.
In fact, Gao Caixia pointed out that after 10 years of development, genome editing technology has become more than just the concept of "a pair of scissors". The base editing and guided editing that evolved to the "2.0 era" can also be a "eraser" or a "pencil".
"If a sequence is a bit much, you can cut it out; if the four letters that make up DNA, ATCG, are wrong, you can erase it like an 'eraser' and write the correct letter with a 'pencil', and the 'pencil' and 'eraser' are not left in the cell." She said metaphorically.
The good news is that at the end of January this year, the Ministry of Agriculture and Rural Affairs formulated and promulgated the "Guidelines for the Safety Evaluation of Gene-Edited Plants for Agriculture (Trial)", which further standardized the safety evaluation and management of agricultural gene-edited plants and promoted the development of biological breeding technology and industry in mainland China. "Encouraged and spurred by this policy, I believe that more genome editing materials will soon enter the field and market on the mainland." Gao Caixia said that the next step will be to carry out the development and promotion of new germplasm resources for wheat powdery mildew.
Related thesis information: DOI: 10.1038/s41586-022-04395-9
Source: Xiao Ke Life