Opening a new door to green pesticides: A new type of fungicide that "kills rice cancer" is born!

Rice blast, also known as "rice cancer", is a devastating fungal disease that causes 3 billion kilograms of grain losses in my country each year. It seriously restricts the stable, high and high yields of my country's staple food crops and threatens my country's food security.

In order to overcome rice blast, Professor Zhang Zhengguang of the College of Plant Protection at Nanjing Agricultural University led his team to fight the pathogen for nearly 30 years. On February 26, 2024, Zhang Zhengguang's team and Professor Xing Weiman's team at Shanghai Normal University published their latest research results in Nature Plants.

They discovered the conservative toxic effector MoErs1, which is unique to rice blast fungus, and revealed its mechanism of suppressing host immunity. The diphenyl ether ester compounds designed based on this mechanism have significant protective effects against rice blast. This study is the first in China and abroad to propose a new concept of creating fungicides using conservative effectors of rice blast fungus as targets.

The reviewers of the paper unanimously agreed that their work is highly innovative and has important implications for finding new strategies to control diseases in the fields of plant-microbe interactions, plants, and agriculture.

**Challenging common sense: ** Will effectors be new targets?

Rice is the main grain crop in my country, and its planting area accounts for about 30% of the total grain crop area in my country. Zhang Zhengguang, co-corresponding author of the paper, said that rice blast, caused by rice blast fungi, can cause a 40% to 50% reduction in rice production in severe cases, or even a total loss of rice, which seriously restricts the stable, high and high yields of rice in my country. At present, the application of fungicides and the use of disease-resistant varieties are the main means of preventing and controlling rice blast.

However, the number of molecular targets for fungicides that can be used to create green pesticides is very limited. According to statistics from the International Fungicide Resistance Action Committee (FRAC), there are only more than 20 targets that can be used for fungicide development so far. Most fungicides are developed for the same target, and 60% of the fungicides widely used in the world only target three targets.

The targets of the fungicides currently used are mostly genes essential for the growth and development of pathogens. The proteins expressed by these genes often control important physiological and biochemical processes for the survival of pathogens. Once destroyed, the pathogens themselves cannot survive.

Zhang Zhengguang explained that the limited number of targets has resulted in the existing drugs having a single target and serious structural homogeneity. Once the key genes of the pathogen mutate, the drug may lose its activity, thus causing a huge risk of drug resistance.

For example, fungicides may only work on specific biological processes, and pathogens can easily develop resistance through mutations in a single mechanism; in addition, a target is acted upon by multiple different fungicides, and pathogens may resist multiple fungicides at the same time by mutating the target, thereby increasing the risk of resistance.

"Therefore, in-depth analysis of the molecular mechanism of the interaction between rice blast fungus and rice, exploring new fungicide targets, and creating green, efficient and low-toxic fungicides are expected to establish new strategies for the green prevention and control of rice blast," said Zhang Zhengguang.

In long-term research, Zhang Zhengguang's team discovered that effector protein is a necessary "weapon" secreted by pathogens to the host plant - rice, which suppresses host immunity and promotes pathogen infection.

During the long-term interaction between plants and pathogens, plants rely on pattern recognition receptors on the cell membrane to perceive pathogen-related pattern molecules and trigger PTI immune responses; in order to suppress the host's PTI immune response, pathogens secrete a large number of effectors into host cells, target important immune components in the host, inhibit the PTI immune response, and cause susceptibility.

In turn, in order to cope with the susceptibility triggered by effectors, plants gradually evolved disease-resistant proteins that specifically recognized effectors, thereby triggering a stronger ETI immune response. The host's ETI exerts a strong selection pressure on pathogens. In order to survive and colonize the host plant, the effectors of the pathogens will continue to mutate and evolve, thereby evading the recognition of the host's disease-resistant proteins and circumventing the ETI immune response of rice. This is also the reason why the resistance of resistant varieties is easily overcome.

It can be seen that most effectors are not conservative and are prone to mutation. Therefore, there is no precedent for developing fungicides using effectors as targets.

Zhang Zhengguang's team was not limited by common sense. "Since effectors are so important in the infection process of bacteria, can we develop new fungicides against rice blast by targeting conservative effectors?" In 2013, Liu Muxing, co-first author of the paper and associate professor of the College of Plant Protection of Nanjing Agricultural University, took on the task of finding new targets for fungicides.

Ten years to uncover the mystery of effectors

"In fact, it is not easy to analyze the function of effector MoErs1," said Liu Muxing.

The secretion of eukaryotic proteins mainly depends on the vesicle transport process, and the secretion of pathogen effectors is also the same. When the relevant genes regulating vesicle transport are knocked out, the process of pathogen secretion of effectors is interrupted, and the infection of rice will not occur.

Zhang Zhengguang's team has accumulated a lot of work on the vesicle transport process of rice blast fungus, and discovered effectors that are secreted and regulated by vesicle transport through extracellular proteomics. In order to find toxic effectors necessary for pathogenicity, they knocked out the identified effector encoding genes one by one, and finally found that the loss of an effector called MoErs1 could interrupt the infection process of the fungus. They realized that this was a very critical effector and might be a breakthrough in their research.

In 2015, their team had already identified the effector MoErs1. "But because there was no special functional domain in its sequence, we were unable to find out its specific function."

In the following eight years, Zhang Zhengguang's team quietly carried out the analysis step by step. "In fact, there is no particularly shocking or interesting story. It is a work that we have worked hard on and invested a lot of time in." Liu Muxing said. In order to further study the function of the effector MoErs1 and uncover its mystery, they first analyzed its polymorphism and found that there was no polymorphism in the hundreds of physiological subspecies of rice blast fungi from all over the world that had been sequenced, indicating that it is highly conservative and not prone to mutation. "This conservatism also determines its importance in the pathogenic process of rice blast fungi." Liu Muxing said.

Since the sequence did not correspond to a special function, they could only find a way to study the three-dimensional structure of the effector. Although this process was very difficult, they finally solved the crystal structure of the effector MoErs1 in collaboration with Xing Weiman's team and found that it is a type of cysteine ​​protease inhibitor that can target the rice cysteine ​​protease OsRD21 and inhibit its enzymatic activity, thereby interfering with the role of OsRD21 in rice immunity.

"Due to the conservatism of MoErs1, it can theoretically be developed and utilized as a target for new fungicides." Zhang Zhengguang said that since conserved effectors are proteins that have been proven to be less prone to mutation during long-term evolution, fungicides developed by looking for conservative effectors as targets are theoretically less likely to develop drug resistance.

New fungicide has been born

Based on the model of the interaction between effectors and the rice immune system, they designed a diphenyl ether ester compound that can competitively bind to the pathogen effector MoErs1 and inhibit its targeting of rice OsRD21, thereby releasing the role of OsRD21 in rice immunity, resisting rice blast infection, and effectively controlling the occurrence of the disease.

"At present, we have completed preliminary experiments on the compound's acute toxicity, triple toxicity and environmental toxicity, and all of them are low toxic," said Liu Muxing.

In September 2023, a field observation meeting on the prevention and control of rice blast by diphenyl ether ester compound FY21001 was held in Taojiang County, Hunan Province. Participants observed the rice blast control efficacy test base in Luoxi Village, Gaoqiao Town, Taojiang County, and Zhang Zhengguang introduced the incidence of rice blast, the application process and the control effect.

The expert group checked the rice blast disease index and field control efficacy of each agent treatment on site, and unanimously agreed that the control effect of diphenyl ether ester compounds was equivalent to that of the control agent tricyclazole.

Bai Lianyang, an academician of the Chinese Academy of Engineering and secretary of the Party Committee of Hunan Academy of Agricultural Sciences, fully affirmed the significance of this work. He evaluated that the diphenyl ether ester compound FY21001 is a new type of high-efficiency and low-toxic fungicide with an original structure developed based on an original target, which breaks through the limitations of traditional fungicide creation. Its field control effect on rice blast is outstanding, and its effect is comparable to that of the existing mainstream pesticides for rice blast control. It is recommended to accelerate the entry into the pesticide registration procedure.

"We have obtained four national authorized invention patents around this compound, which protect the skeleton of the compound. We are currently cooperating with Jiangsu Zhongqi Technology Co., Ltd. to develop it, and we hope to formally submit an application for pesticide registration in the next two years," said Zhang Zhengguang.

Effectors are important weapons for pathogens to attack hosts. Reported research has mainly focused on analyzing the mechanism by which effector proteins inhibit host immunity. Since most effector proteins are polymorphic, there is no precedent for developing fungicides targeting effectors.

This study has expanded people's understanding of fungicide research and development, and created a new strategy for the creation of new fungicides with effectors as targets. At the same time, since effectors are proteins that are unique to fungi and secreted outside the cell, fungicides developed for effectors have the characteristics of low toxicity and low resistance to drug resistance, which meets the development needs of green pesticide creation in my country and opens up a new path for the creation of green pesticides in my country.

Reporter/Xia Wenyan, Correspondent/Xu Tianying