Known as "rice cancer," rice blast disease is a devastating fungal infection that causes annual losses of 3 billion kilograms of grains in China alone, severely impeding the stable, high, and quality yield of our staple crops and threatening food security.
To combat rice blast disease, Professor Zhang Zhengguang from the College of Plant Protection at Nanjing Agricultural University has led a team in a battle against the pathogen for nearly 30 years. On February 26th, Professor Zhang's team, along with Professor Xing Weiman's team from Shanghai Normal University, published their latest research findings in Nature Plants.
They discovered a specific conserved toxic effector, MoErs1, produced by the rice blast pathogen and revealed its mechanism of suppressing host immunity. Compounds based on this mechanism, particularly diphenyl ether esters, exhibited significant efficacy against rice blast disease. This research pioneers a new concept of targeting conserved effectors of the rice blast pathogen to develop fungicides, marking a significant advancement in plant-microbe interactions and strategies for disease control in agriculture.
Reviewers unanimously praised the innovation of their work, recognizing its significant implications for finding new strategies to control diseases in plants and agriculture.
Challenging the Norm: Can Effectors Become New Targets?
Rice is a primary staple crop in China, occupying approximately 30% of the total grain cultivation area. Co-corresponding author Zhang Zhengguang informed Chinese Science Bulletin that rice blast disease caused by the pathogen can lead to yield losses of 40% to 50%, or even complete crop failure, severely restricting stable, high, and quality rice production in China. Currently, the main methods for preventing and controlling rice blast disease involve the application of fungicides and the use of resistant varieties.
Symptoms of rice blast disease in the field. Image provided by the interviewee.
However, the number of molecular targets available for the creation of green pesticides is very limited. According to statistics from the Fungicide Resistance Action Committee (FRAC), there are only over 20 targets available for fungicide development so far. Most fungicides are developed against the same target, with 60% of globally used fungicides targeting only 3 targets.
Currently adopted fungicide targets are mostly essential genes for the growth and development of pathogens. The proteins expressed by these genes often control important physiological and biochemical processes for pathogen survival. Once disrupted, the pathogens themselves cannot survive.
Zhang Zhengguang explained that due to the limited number of targets, existing pesticide action points are singular, and structural homogenization is severe. Once key genes of pathogens mutate, there is a possibility of loss of pesticide activity, thus posing a huge risk of resistance.
For example, fungicides may only act on specific biological processes, and pathogens can easily develop resistance through mutations in a single mechanism. Additionally, a target may be acted upon by multiple different fungicides, and pathogens may develop resistance to multiple fungicides simultaneously by mutating this target, thereby increasing the risk of resistance.
"Therefore, by thoroughly analyzing the molecular mechanism of interaction between rice blast pathogens and rice, exploring new fungicide targets, and developing green, efficient, and low-toxicity fungicides, new strategies for the green control of rice blast disease can be established," Zhang Zhengguang said.
In their long-term research, Zhang Zhengguang's team found that effector proteins are necessary "weapons" for pathogenic fungi to inhibit host immunity and promote fungal infection of host plants like rice.
In the long-term interaction between plants and pathogenic fungi, plants rely on pattern recognition receptors on the cell membrane to perceive pathogen-associated molecular patterns, triggering PTI immune responses. To suppress the host's PTI immune response, pathogens secrete a large number of effectors into host cells, targeting important immune components in the host to suppress PTI immune responses and induce susceptibility.
In response, to counteract effector-triggered susceptibility, plants gradually evolve resistance proteins that specifically recognize effectors, thereby triggering a stronger ETI immune response. The host's ETI exerts strong selection pressure on pathogens. To survive and colonize the host plant, pathogenic fungi's effectors continuously mutate and evolve to evade recognition by host resistance proteins, circumventing rice's ETI immune response. This is also why resistance in susceptible varieties is easily overcome.
It can be seen that effectors are mostly non-conserved and prone to mutation. Therefore, there has been no precedent for developing fungicides targeting effectors.
Zhang Zhengguang's team was not limited by common sense. "Since effectors are so important in the process of pathogen infection, can we develop new rice blast fungicides targeting conserved effectors?" In 2013, Liu Muxing, co-first author of the paper and associate professor of plant protection at Nanjing Agricultural University, took over the task of finding new fungicide targets.
Ten years just to uncover the mystery of effectors
"In fact, deciphering the function of the effector MoErs1 was not easy at all," recalled Liu Muxing.
The secretion of eukaryotic proteins mainly relies on vesicular transport processes, and the secretion of pathogenic effector proteins is also the same. When genes regulating vesicle transport are knocked out, the process of pathogen secreting effectors is interrupted, and infection of rice will not occur.
Zhang Zhengguang's team has accumulated a lot of work on the vesicular transport process of rice blast fungus and identified effectors regulated by vesicle transport through extracellular proteomics. In order to find toxic effectors necessary for pathogen virulence, they knocked out each identified effector-encoding gene one by one and finally found that the absence of an effector called MoErs1 could interrupt the infection process of the pathogen. They realized that this was a very critical effector, perhaps the breakthrough in their research.
In 2015, their team had already identified the effector MoErs1. "But because it lacks specific functional domains in its sequence, we have been unable to determine its specific function."
In the following eight years, Zhang Zhengguang's team silently carried out step-by-step analysis work. "Actually, there are no particularly shocking or interesting stories. It's just a work that we persisted in and invested a lot of time in," Liu Muxing said.
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 among hundreds of physiologic races of rice blast fungi sequenced from around the world, indicating its high conservatism and low propensity for variation. "This conservatism also determines its importance in the pathogenesis of rice blast fungi," Liu Muxing said.
Since the sequence does not correspond to specific functions, they could only find ways to study the three-dimensional structure of the effector. Although this process was very difficult, they finally, with the cooperation of Xing Weiman's team, resolved the crystal structure of the effector MoErs1, finding that it is a type of cysteine protease inhibitor that targets the rice cysteine protease OsRD21 and inhibits its enzyme activity, thereby interfering with the role of OsRD21 in rice immunity.
The discovery of the inhibitory effect of the rice blast fungus effector MoErs1 on rice immunity and the creation strategy for the first targeted MoErs1 inhibitor.
"Due to the conservatism of MoErs1, it could theoretically serve as a novel target for fungicide development," said Zhang Zhengguang. "Because conservative effectors are proteins that have been proven not to mutate easily during long-term evolution, finding conservative effectors as targets for fungicide development theoretically reduces the likelihood of resistance."
A novel fungicide has emerged.
Based on a model of the interaction between effectors and the rice immune system, they designed a class of diphenyl ether compounds. These compounds competitively bind to the pathogen effector MoErs1, inhibiting its targeting of OsRD21 in rice. This releases OsRD21's role in rice immunity, enabling resistance against blast fungus infection and effectively controlling disease occurrence.
"We have completed preliminary experiments on acute toxicity, three toxicity, and environmental toxicity of this compound, and all show low toxicity," said Liu Muxing.
In September 2023, a field observation meeting on the control of blast disease in rice with the diphenyl ether compound FY21001 was held in Taojiang County, Hunan Province. Attendees observed the blast disease control efficacy trial base in Luoxi Village, Gaoqiao Town, Taojiang County. Zhang Zhengguang introduced the disease incidence, pesticide application process, and control effect of blast disease.
An expert group inspected the blast disease index and field efficacy of various pesticide treatments and unanimously agreed that the diphenyl ether compound performed as effectively as the control pesticide, tricyclazole.
Ba Lianyang, academician of the Chinese Academy of Engineering and secretary of the Party Committee of Hunan Academy of Agricultural Sciences, fully acknowledged the significance of this work. He praised FY21001 as a novel, highly efficient, low-toxicity fungicide based on original target development and a novel structure, breaking through the limitations of traditional fungicide development. Its outstanding field efficacy against blast disease is comparable to mainstream pesticides for blast disease control, suggesting expedited entry into pesticide registration procedures.
"Around this compound, we have obtained four national authorized invention patents to protect its structure. Currently, we are collaborating with Jiangsu Zhongqi Technology Co., Ltd., and expect to formally submit pesticide registration applications within the next two years," Zhang Zhengguang said.
Effectors are crucial weapons for pathogens attacking hosts. Previous research mainly focused on deciphering how effector proteins inhibit host immunity. However, as most effector proteins are highly polymorphic, there have been no precedents for developing fungicides targeting effectors.
This study expands our understanding of fungicide development and establishes a new strategy for creating fungicides targeting effectors. Furthermore, since effectors are specific to fungi and are secreted into the extracellular space, fungicides targeting effectors exhibit characteristics such as low toxicity and reduced likelihood of resistance. This aligns with the development needs of green pesticide creation in China and opens up new pathways for green pesticide development.
Paper reference: Nature Article
