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On January 13, 2021, Professor Wang Shiping’s group from the State Key Laboratory of Crop Genetic Improvement of Huazhong Agricultural University published a report on Molecular Plant entitled "Pathogen-inducible OsMPKK10.
2-OsMPK6 cascade phosphorylates the Raf-like kinase OsEDR1 and inhibits its The research paper "scaffold function to promote rice disease resistance" reveals the mutual regulation mechanism between a Raf-like protein kinase and mitogen-activated protein kinase (MAPK) signal cascade in rice immune response.
Understanding the function and mechanism of Raf protein kinases provides a new perspective.
Many physiological activities of plants, including immune response, are involved in the MAPK signaling cascade.
A complete MAPK signal cascade includes three kinases, MAPKKK (MAPK kinase), MAPKK (MAPK kinase) and MAPK.
The MAPK signal cascade transmits extracellular signals into the cell through sequential phosphorylation.
MAPKKK is phosphorylated in response to external signals, and then phosphorylates the serine (S)/threonine in the specific motif of MAPKK (S/T-X3-5-S/T; X represents any amino acid, and the number represents the number of amino acids in the interval) Acid (T), thereby activating MAPKK; activated MAPKK then phosphorylates threonine and tyrosine (Y) in a specific motif (TXY) of MAPK, thereby activating MAPK.
The activated MAPK phosphorylates specific substrates (including transcription factors, enzymes, etc.
), so that cells respond to external signals.Compared with yeast and animals, the number of MAPKKK in plants is relatively large, which can be simply divided into two categories: MEKK and Raf.
Among them, the number of Raf types is relatively large.
For example, there are 75 kinases similar to MAPKKK in rice, and 43 of Raf.
The MAPKKK of the MEKK class regulates the MAPK signal cascade in a normal way, that is, directly phosphorylates and activates MAPKK, which in turn activates MAPK.
For example, there are at least two complete MAPK signal cascades in Arabidopsis that are involved in regulating the immune response, namely MEKK1-MKK1/MKK2-MPK4 and MAPKKK3/MAPKKK5-MKK4/MKK5-MPK3/MPK6.
Among them, MEKK1 and MAPKKK3/MAPKKK5 belong to MEKK kinases.
After they are phosphorylated by cytoplasmic receptor kinases, they directly phosphorylate and activate the corresponding MAPKK.
Raf MAPKKK is also involved in the regulation of plant MAPK signaling cascade.
There are two well-known Raf MAPKKKs in Arabidopsis, CTR1 and EDR1.
CTR1 negatively regulates the MKK9-MPK3/MPK6 signal cascade, which in turn regulates the ethylene signal pathway in plants.
EDR1 negatively regulates the MKK4/MKK5-MPK3/MPK6 signaling cascade, which in turn regulates the innate immunity of plants.
Among them, the mechanism of EDR1 is relatively clear.
EDR1 physically interacts with MKK4/MKK5 through its amino terminus, and negatively regulates the protein accumulation of MKK4/MKK5; at the same time, EDR1 inhibits the phosphorylation of an E3 ubiquitin ligase, thereby inhibiting the protein accumulation of MKK4/MKK5.
From these results, it can be seen that in the MAPK signal cascade of plants, Raf MAPKKKs act differently from MEKKs.
The research started with a MAPKK of rice, namely OsMPKK 10.
2.The previous results showed that the OsMPKK10.
2-OsMPK6 signal cascade positively regulates the resistance of rice to bacterial leaf leaf spot (Xanthomonas oryzae pv.
oryzicola, Xoc).
OsMPKK10.
2 is a structurally atypical MAPKK.
The potential phosphorylated motif of MAPKKK in its protein sequence is missing.
Therefore, the complete activation of OsMPKK10.
2 may require phosphorylation at other sites.
The author used mass spectrometry to identify the phosphorylation sites on OsMPKK10.
2 after Xoc inoculation in rice.
One of the sites, the motif of the S at position 304, is consistent with the substrate characteristics of AGC kinase.
S304 was mutated to aspartic acid (D) to simulate the phosphorylation state of this site, and then this form of OsMPKK10.
2 was overexpressed in the wild type, and it was found that the transgenic rice showed a disease-resistant phenotype to Xoc, and at the same time in vivo OsMPK6 is activated, and the expression of OsWRKY45 (substrate of OsMPK6) also increases, and visible cell death (called pseudo-spots) appears on rice leaves, and a large amount of H2O2 is also accumulated.
These results indicate that phosphorylation at S304 is very important for activating OsMPKK10.
2 and subsequent immune responses.
In another parallel experiment, the authors found that OsEDR1 (the homologous protein of Arabidopsis EDR1) physically interacts with OsMPKK10.
2, but the OsEDR1 protein expressed in eukaryotic cells does not phosphorylate OsMPKK10.
2 in vitro .
The published work of the research group has shown that OsEDR1 negatively regulates rice resistance to bacterial diseases, and that the osedr1 mutant leaves pseudo-spots.
Therefore, in the immune response of rice, OsEDR1 and OsMPKK10.
2 may be located in the same signal path. Biochemical analysis found that compared with the wild type, the phosphorylation of OsMPKK10.
2 S304 site was enhanced in the osedr1 mutant, and the activity of OsMPKK10.
2 was also enhanced, correspondingly the activation of OsMPK6 and the expression of OsWRKY45 were also enhanced.
These changes were inoculated.
It is more obvious after Xoc; while in the complementary plants of the osedr1 mutant, the phosphorylation and kinase activity of OsMPKK10.
2, the activation of OsMPK6 and the expression of OsWRKY45 are similar to those in the wild type.
Using CRISPR/Cas9 technology to knock out OsMPKK10.
2 in the osedr1 mutant, the double mutant showed a disease-resistant phenotype similar to that of the wild type, and the pseudo-spot phenomenon was weakened compared with the osedr1 mutant; and the osedr1/osmpk6 double mutation The body showed a susceptible phenotype to Xoc, and there was no false disease spots on the leaves.
These results indicate that OsEDR1 inhibits the activation of the OsMPKK10.
2-OsMPK6 signaling cascade.
It also indicates that OsMPKK10.
2 is not the only MAPKK acting downstream of OsEDR1, and also indicates that cell death and immune response are not completely coupled.
The protein sequence of OsEDR1 contains a large number of recognition sites for MAPK, and the author speculates that OsEDR1 will be phosphorylated by OsMPK6.
Through in vitro phosphorylation combined with mass spectrometry analysis, it was found that the S861 site in the kinase region of OsEDR1 is the main phosphorylation site of OsMPK6.
Further analysis found that in wild-type rice, the phosphorylation of this site occurred after Xoc infection.
At the same time, the phosphorylation of this site also exists in OsMPKK10.
2 overexpression plants, and is dependent on OsMPK6.
When OsMPKK10.
2 S304 site is phosphorylated, OsEDR1 S861 site phosphorylation will also occur.
These results indicate that OsMPKK10.
2-OsMPK6 phosphorylates OsEDR1 after being activated by pathogenic bacteria.
The author mutated the S861 site of OsEDR1 to alanine (A) so that the site could not be phosphorylated.
When this form of OsEDR1 (OsEDR1S861A) was transferred into the osedr1 mutant, the transgenic plants showed a susceptible phenotype to Xoc, and there were no false spots on the leaves.
At the same time, the activation of the OsMPKK10.
2-OsMPK6 signal cascade was also affected.
Inhibition.
Further research found that OsEDR1S861A can still interact physically with OsMPKK10.
2.
In addition, after Xoc infection, OsEDR1 will be degraded, while OsEDR1S861A is not easily degraded, indicating that the phosphorylation of S861 site promotes the degradation of OsEDR1 induced by pathogens.
The authors infer from this (Figure 1) that OsEDR1 inhibits the activation of OsMPKK10.
2 by interacting with a part of OsMPKK10.
2; when pathogens invade, the OsMPKK10.
2 S304 site is phosphorylated by an unknown kinase to activate OsMPK6.
The latter phosphorylates the S861 site of OsEDR1, promotes the degradation of OsEDR1, and releases more OsMPKK10.
2, which amplifies the activation of the OsMPKK10.
2-OsMPK6 signaling cascade, thereby enhancing the immune response of rice.
These results indicate that OsEDR1 takes the form of a scaffold protein involved in the regulation of the OsMPKK10.
2-OsMPK6 signaling cascade.
Figure 1.
The inter-regulatory relationship between OsEDR1 and OsMPKK10.
2-OsMPK6 signaling cascade in rice immune response.
The above-mentioned molecular mechanism is a universal regulation mechanism in eukaryotes.
In yeast, Ste5 is a scaffolding protein that regulates the MAPK signal cascade Ste11-Ste7-Fus3; Fus3 (a MAPK) phosphorylates Ste5 through feedback, reducing the signal output of the MAPK signal cascade.
In mammalian cells, KSR1 is a scaffolding protein that regulates the MAPK signaling cascade Raf-MEK-ERK; ERK (a MAPK) phosphorylates KSR1 through feedback, cutting off the activation of the MAPK signaling cascade.
In Arabidopsis, after pathogen infection, the two MAPK signal cascades MAPKKK3/5-MKK4/5-MPK3/6 and MEKK1-MKK1/2-MPK4 in MPK6 and MPK4 feedback phosphorylation of MAPKKK5 and MEKK1, respectively.
Enhance the activation of MAPK.
Therefore, the results of this study once again show that the way that MAPK regulates signal output by phosphorylation of scaffold proteins or MAPKKK in the signal cascade is conserved in eukaryotes.
Of course, there are many important issues that remain unresolved.
For example, what is the significance of the kinase activity of Raf MAPKKK? Does the kinase activity change after it is phosphorylated by downstream MAPK? Some important studies have found that Raf-like MAPKKK in plants phosphorylates SnRK2 and regulates the abiotic stress response of plants, which seems to imply that the emergence of Raf-like MAPKKK in plants is to adapt to adversity stress.
But how does Raf MAPKKK recognize non-MAPKK substrates? How is the activation of its kinase activity regulated? These are worthy of further exploration.
Professor Wang Shiping of Huazhong Agricultural University is the corresponding author of the paper, and Ma Haigang, a postdoctoral fellow and Research Associate, is the first author and co-corresponding author of the paper.
The research was funded by the National Natural Science Foundation of China and the Youth Science Foundation, as well as the Independent Science and Technology Innovation Fund of Huazhong Agricultural University.
2-OsMPK6 cascade phosphorylates the Raf-like kinase OsEDR1 and inhibits its The research paper "scaffold function to promote rice disease resistance" reveals the mutual regulation mechanism between a Raf-like protein kinase and mitogen-activated protein kinase (MAPK) signal cascade in rice immune response.
Understanding the function and mechanism of Raf protein kinases provides a new perspective.
Many physiological activities of plants, including immune response, are involved in the MAPK signaling cascade.
A complete MAPK signal cascade includes three kinases, MAPKKK (MAPK kinase), MAPKK (MAPK kinase) and MAPK.
The MAPK signal cascade transmits extracellular signals into the cell through sequential phosphorylation.
MAPKKK is phosphorylated in response to external signals, and then phosphorylates the serine (S)/threonine in the specific motif of MAPKK (S/T-X3-5-S/T; X represents any amino acid, and the number represents the number of amino acids in the interval) Acid (T), thereby activating MAPKK; activated MAPKK then phosphorylates threonine and tyrosine (Y) in a specific motif (TXY) of MAPK, thereby activating MAPK.
The activated MAPK phosphorylates specific substrates (including transcription factors, enzymes, etc.
), so that cells respond to external signals.Compared with yeast and animals, the number of MAPKKK in plants is relatively large, which can be simply divided into two categories: MEKK and Raf.
Among them, the number of Raf types is relatively large.
For example, there are 75 kinases similar to MAPKKK in rice, and 43 of Raf.
The MAPKKK of the MEKK class regulates the MAPK signal cascade in a normal way, that is, directly phosphorylates and activates MAPKK, which in turn activates MAPK.
For example, there are at least two complete MAPK signal cascades in Arabidopsis that are involved in regulating the immune response, namely MEKK1-MKK1/MKK2-MPK4 and MAPKKK3/MAPKKK5-MKK4/MKK5-MPK3/MPK6.
Among them, MEKK1 and MAPKKK3/MAPKKK5 belong to MEKK kinases.
After they are phosphorylated by cytoplasmic receptor kinases, they directly phosphorylate and activate the corresponding MAPKK.
Raf MAPKKK is also involved in the regulation of plant MAPK signaling cascade.
There are two well-known Raf MAPKKKs in Arabidopsis, CTR1 and EDR1.
CTR1 negatively regulates the MKK9-MPK3/MPK6 signal cascade, which in turn regulates the ethylene signal pathway in plants.
EDR1 negatively regulates the MKK4/MKK5-MPK3/MPK6 signaling cascade, which in turn regulates the innate immunity of plants.
Among them, the mechanism of EDR1 is relatively clear.
EDR1 physically interacts with MKK4/MKK5 through its amino terminus, and negatively regulates the protein accumulation of MKK4/MKK5; at the same time, EDR1 inhibits the phosphorylation of an E3 ubiquitin ligase, thereby inhibiting the protein accumulation of MKK4/MKK5.
From these results, it can be seen that in the MAPK signal cascade of plants, Raf MAPKKKs act differently from MEKKs.
The research started with a MAPKK of rice, namely OsMPKK 10.
2.The previous results showed that the OsMPKK10.
2-OsMPK6 signal cascade positively regulates the resistance of rice to bacterial leaf leaf spot (Xanthomonas oryzae pv.
oryzicola, Xoc).
OsMPKK10.
2 is a structurally atypical MAPKK.
The potential phosphorylated motif of MAPKKK in its protein sequence is missing.
Therefore, the complete activation of OsMPKK10.
2 may require phosphorylation at other sites.
The author used mass spectrometry to identify the phosphorylation sites on OsMPKK10.
2 after Xoc inoculation in rice.
One of the sites, the motif of the S at position 304, is consistent with the substrate characteristics of AGC kinase.
S304 was mutated to aspartic acid (D) to simulate the phosphorylation state of this site, and then this form of OsMPKK10.
2 was overexpressed in the wild type, and it was found that the transgenic rice showed a disease-resistant phenotype to Xoc, and at the same time in vivo OsMPK6 is activated, and the expression of OsWRKY45 (substrate of OsMPK6) also increases, and visible cell death (called pseudo-spots) appears on rice leaves, and a large amount of H2O2 is also accumulated.
These results indicate that phosphorylation at S304 is very important for activating OsMPKK10.
2 and subsequent immune responses.
In another parallel experiment, the authors found that OsEDR1 (the homologous protein of Arabidopsis EDR1) physically interacts with OsMPKK10.
2, but the OsEDR1 protein expressed in eukaryotic cells does not phosphorylate OsMPKK10.
2 in vitro .
The published work of the research group has shown that OsEDR1 negatively regulates rice resistance to bacterial diseases, and that the osedr1 mutant leaves pseudo-spots.
Therefore, in the immune response of rice, OsEDR1 and OsMPKK10.
2 may be located in the same signal path. Biochemical analysis found that compared with the wild type, the phosphorylation of OsMPKK10.
2 S304 site was enhanced in the osedr1 mutant, and the activity of OsMPKK10.
2 was also enhanced, correspondingly the activation of OsMPK6 and the expression of OsWRKY45 were also enhanced.
These changes were inoculated.
It is more obvious after Xoc; while in the complementary plants of the osedr1 mutant, the phosphorylation and kinase activity of OsMPKK10.
2, the activation of OsMPK6 and the expression of OsWRKY45 are similar to those in the wild type.
Using CRISPR/Cas9 technology to knock out OsMPKK10.
2 in the osedr1 mutant, the double mutant showed a disease-resistant phenotype similar to that of the wild type, and the pseudo-spot phenomenon was weakened compared with the osedr1 mutant; and the osedr1/osmpk6 double mutation The body showed a susceptible phenotype to Xoc, and there was no false disease spots on the leaves.
These results indicate that OsEDR1 inhibits the activation of the OsMPKK10.
2-OsMPK6 signaling cascade.
It also indicates that OsMPKK10.
2 is not the only MAPKK acting downstream of OsEDR1, and also indicates that cell death and immune response are not completely coupled.
The protein sequence of OsEDR1 contains a large number of recognition sites for MAPK, and the author speculates that OsEDR1 will be phosphorylated by OsMPK6.
Through in vitro phosphorylation combined with mass spectrometry analysis, it was found that the S861 site in the kinase region of OsEDR1 is the main phosphorylation site of OsMPK6.
Further analysis found that in wild-type rice, the phosphorylation of this site occurred after Xoc infection.
At the same time, the phosphorylation of this site also exists in OsMPKK10.
2 overexpression plants, and is dependent on OsMPK6.
When OsMPKK10.
2 S304 site is phosphorylated, OsEDR1 S861 site phosphorylation will also occur.
These results indicate that OsMPKK10.
2-OsMPK6 phosphorylates OsEDR1 after being activated by pathogenic bacteria.
The author mutated the S861 site of OsEDR1 to alanine (A) so that the site could not be phosphorylated.
When this form of OsEDR1 (OsEDR1S861A) was transferred into the osedr1 mutant, the transgenic plants showed a susceptible phenotype to Xoc, and there were no false spots on the leaves.
At the same time, the activation of the OsMPKK10.
2-OsMPK6 signal cascade was also affected.
Inhibition.
Further research found that OsEDR1S861A can still interact physically with OsMPKK10.
2.
In addition, after Xoc infection, OsEDR1 will be degraded, while OsEDR1S861A is not easily degraded, indicating that the phosphorylation of S861 site promotes the degradation of OsEDR1 induced by pathogens.
The authors infer from this (Figure 1) that OsEDR1 inhibits the activation of OsMPKK10.
2 by interacting with a part of OsMPKK10.
2; when pathogens invade, the OsMPKK10.
2 S304 site is phosphorylated by an unknown kinase to activate OsMPK6.
The latter phosphorylates the S861 site of OsEDR1, promotes the degradation of OsEDR1, and releases more OsMPKK10.
2, which amplifies the activation of the OsMPKK10.
2-OsMPK6 signaling cascade, thereby enhancing the immune response of rice.
These results indicate that OsEDR1 takes the form of a scaffold protein involved in the regulation of the OsMPKK10.
2-OsMPK6 signaling cascade.
Figure 1.
The inter-regulatory relationship between OsEDR1 and OsMPKK10.
2-OsMPK6 signaling cascade in rice immune response.
The above-mentioned molecular mechanism is a universal regulation mechanism in eukaryotes.
In yeast, Ste5 is a scaffolding protein that regulates the MAPK signal cascade Ste11-Ste7-Fus3; Fus3 (a MAPK) phosphorylates Ste5 through feedback, reducing the signal output of the MAPK signal cascade.
In mammalian cells, KSR1 is a scaffolding protein that regulates the MAPK signaling cascade Raf-MEK-ERK; ERK (a MAPK) phosphorylates KSR1 through feedback, cutting off the activation of the MAPK signaling cascade.
In Arabidopsis, after pathogen infection, the two MAPK signal cascades MAPKKK3/5-MKK4/5-MPK3/6 and MEKK1-MKK1/2-MPK4 in MPK6 and MPK4 feedback phosphorylation of MAPKKK5 and MEKK1, respectively.
Enhance the activation of MAPK.
Therefore, the results of this study once again show that the way that MAPK regulates signal output by phosphorylation of scaffold proteins or MAPKKK in the signal cascade is conserved in eukaryotes.
Of course, there are many important issues that remain unresolved.
For example, what is the significance of the kinase activity of Raf MAPKKK? Does the kinase activity change after it is phosphorylated by downstream MAPK? Some important studies have found that Raf-like MAPKKK in plants phosphorylates SnRK2 and regulates the abiotic stress response of plants, which seems to imply that the emergence of Raf-like MAPKKK in plants is to adapt to adversity stress.
But how does Raf MAPKKK recognize non-MAPKK substrates? How is the activation of its kinase activity regulated? These are worthy of further exploration.
Professor Wang Shiping of Huazhong Agricultural University is the corresponding author of the paper, and Ma Haigang, a postdoctoral fellow and Research Associate, is the first author and co-corresponding author of the paper.
The research was funded by the National Natural Science Foundation of China and the Youth Science Foundation, as well as the Independent Science and Technology Innovation Fund of Huazhong Agricultural University.
