Role of lysine residues of the Magnaporthe oryzae effector AvrPiz-t in effector- and PAMP-triggered immunity

Magnaporthe oryzae is an important fungal pathogen of both rice and wheat. However, how M. oryzae effectors modulate plant immunity is not fully understood. Previous studies have shown that the M. oryzae effector AvrPiz-t targets the host ubiquitin-proteasome system to manipulate plant defence. In return, two rice ubiquitin E3 ligases, APIP6 and APIP10, ubiquitinate AvrPiz-t for degradation. To determine how lysine residues contribute to the stability and function of AvrPiz-t, we generated double (K1,2R-AvrPiz-t), triple (K1,2,3R-AvrPiz-t) and lysine-free (LF-AvrPiz-t) mutants by mutating lysines into arginines in AvrPiz-t. LF-AvrPiz-t showed the highest protein accumulation when transiently expressed in rice protoplasts. When co-expressed with APIP10 in Nicotiana benthamiana, LF-AvrPiz-t was more stable than AvrPiz-t and was less able to degrade APIP10. The avirulence of LF-AvrPiz-t on Piz-t:HA plants was less than that of AvrPiz-t, which led to resistance reduction and lower accumulation of the Piz-t:HA protein after inoculation with the LF-AvrPiz-t-carrying isolate. Chitin- and flg22-induced production of reactive oxygen species (ROS) was higher in LF-AvrPiz-t than in AvrPiz-t transgenic plants. In addition, LF-AvrPiz-t transgenic plants were less susceptible than AvrPiz-t transgenic plants to a virulent isolate. Furthermore, both AvrPiz-t and LF-AvrPiz-t interacted with OsRac1, but the suppression of OsRac1-mediated ROS generation by LF-AvrPiz-t was significantly lower than that by AvrPiz-t. Together, these results suggest that the lysine residues of AvrPiz-t are required for its avirulence and virulence functions in rice.     

The E3 Ligase APIP10 Connects the Effector AvrPiz-t to the NLR Receptor Piz-t in Rice

Although nucleotide-binding domain, leucine-rich repeat (NLR) proteins are the major immune receptors in plants, the mechanism that controls their activation and immune signaling remains elusive. Here, we report that the avirulence effector AvrPiz-t from Magnaporthe oryzae targets the rice E3 ligase APIP10 for degradation, but that APIP10, in return, ubiquitinates AvrPiz-t and thereby causes its degradation. Silencing of APIP10 in the non-Piz-t background compromises the basal defense against M. oryzae. Conversely, silencing of APIP10 in the Piz-t background causes cell death, significant accumulation of Piz-t, and enhanced resistance to M. oryzae, suggesting that APIP10 is a negative regulator of Piz-t. We show that APIP10 promotes degradation of Piz-t via the 26S proteasome system. Furthermore, we demonstrate that AvrPiz-t stabilizes Piz-t during M. oryzae infection. Together, our results show that APIP10 is a novel E3 ligase that functionally connects the fungal effector AvrPiz-t to its NLR receptor Piz-t in rice.     

The Magnaporthe oryzae Effector AvrPiz-t Targets the RING E3 Ubiquitin Ligase APIP6 to Suppress Pathogen-Associated Molecular Pattern–Triggered Immunity in Rice

Although the functions of a few effector proteins produced by bacterial and oomycete plant pathogens have been elucidated in recent years, information for the vast majority of pathogen effectors is still lacking, particularly for those of plant-pathogenic fungi. Here, we show that the avirulence effector AvrPiz-t from the rice blast fungus Magnaporthe oryzae preferentially accumulates in the specialized structure called the biotrophic interfacial complex and is then translocated into rice (Oryza sativa) cells. Ectopic expression of AvrPiz-t in transgenic rice suppresses the flg22- and chitin-induced generation of reactive oxygen species (ROS) and enhances susceptibility to M. oryzae, indicating that AvrPiz-t functions to suppress pathogen-associated molecular pattern (PAMP)-triggered immunity in rice. Interaction assays show that AvrPiz-t suppresses the ubiquitin ligase activity of the rice RING E3 ubiquitin ligase APIP6 and that, in return, APIP6 ubiquitinates AvrPiz-t in vitro. Interestingly, agroinfection assays reveal that AvrPiz-t and AvrPiz-t Interacting Protein 6 (APIP6) are both degraded when coexpressed in Nicotiana benthamiana. Silencing of APIP6 in transgenic rice leads to a significant reduction of flg22-induced ROS generation, suppression of defense-related gene expression, and enhanced susceptibility of rice plants to M. oryzae. Taken together, our results reveal a mechanism in which a fungal effector targets the host ubiquitin proteasome system for the suppression of PAMP-triggered immunity in plants.     

The DnaJ protein OsDjA6 negatively regulates rice innate immunity to the blast fungus Magnaporthe oryzae

Rice blast, caused by Magnaporthe oryzae (synonym: Pyricularia oryzae), severely reduces rice production and grain quality. The molecular mechanism of rice resistance to M. oryzae is not fully understood. In this study, we identified a chaperone DnaJ protein, OsDjA6, which is involved in basal resistance to M. oryzae in rice. The OsDjA6 protein is distributed in the entire rice cell. The expression of OsDjA6 is significantly induced in rice after infection with a compatible isolate. Silencing of OsDjA6 in transgenic rice enhances resistance to M. oryzae and also results in an increased burst of reactive oxygen species after flg22 and chitin treatments. In addition, the expression levels of WRKY45, NPR1 and PR5 are increased in OsDjA6 RNAi plants, indicating that OsDjA6 may mediate resistance by affecting the salicylic acid pathway. Finally, we found that OsDjA6 interacts directly with the E3 ligase OsZFP1 in vitro and in vivo. These results suggest that the DnaJ protein OsDjA6 negatively regulates rice innate immunity, probably via the ubiquitination proteasome degradation pathway.     

A rice gene encoding glycosyl hydrolase plays contrasting roles in immunity depending on the type of pathogens

Because pathogens use diverse infection strategies, plants cannot use one-size-fits-all defence and modulate defence responses based on the nature of pathogens and pathogenicity mechanism. Here, we report that a rice glycoside hydrolase (GH) plays contrasting roles in defence depending on whether a pathogen is hemibiotrophic or necrotrophic. The Arabidopsis thaliana MORE1 (M agnaporthe o ryzae re sistance 1) gene, encoding a member of the GH10 family, is needed for resistance against M. oryzae and Alternaria brassicicola, a fungal pathogen infecting A. thaliana as a necrotroph. Among 13 rice genes homologous to MORE1, 11 genes were induced during the biotrophic or necrotrophic stage of infection by M. oryzae. CRISPR/Cas9-assisted disruption of one of them (OsMORE1a) enhanced resistance against hemibiotrophic pathogens M. oryzae and Xanthomonas oryzae pv. oryzae but increased susceptibility to Cochliobolus miyabeanus, a necrotrophic fungus, suggesting that OsMORE1a acts as a double-edged sword depending on the mode of infection (hemibiotrophic vs. necrotrophic). We characterized molecular and cellular changes caused by the loss of MORE1 and OsMORE1a to understand how these genes participate in modulating defence responses. Although the underlying mechanism of action remains unknown, both genes appear to affect the expression of many defence-related genes. Expression patterns of the GH10 family genes in A. thaliana and rice suggest that other members also participate in pathogen defence.     

2,6‐Dimethoxy‐1,4‐Benzoquinone Enhances Resistance Against the Rice Blast Fungus Magnaporthe oryzae

We investigated the effect of 2,6‐dimethoxy‐1,4‐benzoquinone (DMBQ) on induced resistance to Magnaporthe oryzae in rice. DMBQ concentrations greater than 50 μg/ml inhibited spore germination and appressorium formation in M. oryzae. When rice leaves pretreated with 10 μg/ml DMBQ, which did not show antifungal activity against spore germination and appressorium formation of M. oryzae, were inoculated with M. oryzae spores 5 days after DMBQ pretreatment, blast lesion formation was inhibited compared with control leaves pretreated with distilled water. In addition, infection‐inhibiting activity against M. oryzae was significantly enhanced in rice leaf sheaths pretreated with 10 μg/ml DMBQ. H₂O₂ generation was observed in rice leaves pretreated with DMBQ, and PAL, POX, CHS and PR10a were significantly expressed in these leaves. These results suggested that DMBQ can protect rice from blast disease caused by M. oryzae.     

Rapid and cost-effective screening of CRISPR/Cas9-induced mutants by DNA-guided Argonaute nuclease

Objective

With the widespread application of CRISPR/Cas9 gene editing technology, new methods are needed to screen mutants quickly and effectively. Here, we aimed to develop a simple and cost-effective method to screen CRISPR/Cas9-induced mutants.

Result

We report a novel method to identify CRISPR/Cas9-induced mutants through a DNA-guided Argonaute nuclease derived from the archaeon Pyrococcus furiosus. We demonstrated that the Pyrococcus furiosus Argonaute (PfAgo)-based method could distinguish among biallelic mutants, monoallelic mutants and wild type (WT). Furthermore, this method was able to identify 1 bp indel mutations.

Conclusion

The PfAgo-based method is simple to implement and can be applied to screen biallelic mutants and mosaic mutants generated by CRISPR-Cas9 or other kinds of gene editing tools.

     

Engineering a peptide aptamer to target calmodulin for the inhibition of Magnaporthe oryzae

To develop an antimicrobial agent for preventing the devasting damage caused by rice blast, a novel peptide aptamer was identified to interact with calmodulin (CaM) for the inhibition of the spore development in the pathogen Magnaporthe oryzae. A peptide aptamer designated as SNP-D4, consisted of the scaffold protein Staphylococcus aureus nuclease (SN) and an exposed surface loop of 16 random amino acids, was screened from the constructed peptide aptamer libraries by bacterial two-hybrid system using CaM of M. oryzae as the bait. The preliminary inhibition in the sporulation development was observed after treating with the crude extracts expressing SNP-D4. The inhibition efficacies of the purified SNP-D4 were quantified at the stages of conidial germination, germ tube elongation, and appressorium formation in M. oryzae. The binding affinity analysis revealed that SNP-D4 interacted with CaM at a dissociation constant (K_d) of about 20 μM. Moreover, the N-terminus of CaM was identified as the key binding region.     

Generation of ‘designer erythroblasts’ lacking one or more blood group systems from CRISPR/Cas9 gene-edited human-induced pluripotent stem cells

Despite the recent advancements in transfusion medicine, red blood cell (RBC) alloimmunization remains a challenge for multiparous women and chronically transfused patients. At times, diagnostic laboratories depend on difficult-to-procure rare reagent RBCs for the identification of different alloantibodies in such subjects. We have addressed this issue by developing erythroblasts with custom phenotypes (Rh null, GPB null and Kx null/Kell low) using CRISPR/Cas9 gene-editing of a human induced pluripotent stem cell (hiPSC) parent line (OT1-1) for the blood group system genes: RHAG, GYPB and XK. Guide RNAs were cloned into Cas9-puromycin expression vector and transfected into OT1-1. Genotyping was performed to select puromycin-resistant hiPSC KOs. CRISPR/Cas9 gene-editing resulted in the successful generation of three KO lines, RHAG KO, GYPB KO and XK KO. The OT1-1 cell line, as well as the three KO hiPSC lines, were differentiated into CD34⁺CD41⁺CD235ab⁺ hematopoietic progenitor cells (HPCs) and subsequently to erythroblasts. Native OT1-1 erythroblasts were positive for the expression of Rh, MNS, Kell and H blood group systems. Differentiation of RHAG KO, GYPB KO and XK KO resulted in the formation of Rh null, GPB null and Kx null/Kell low erythroblasts, respectively. OT1-1 as well as the three KO erythroblasts remained positive for RBC markers—CD71 and BAND3. Erythroblasts were mostly at the polychromatic/ orthochromatic stage of differentiation. Up to ~400-fold increase in erythroblasts derived from HPCs was observed. The availability of custom erythroblasts generated from CRISPR/Cas9 gene-edited hiPSC should be a useful addition to the tools currently used for the detection of clinically important red cell alloantibodies.     

Proteomic analysis of rice mutants susceptible to Magnaporthe oryzae

To identify genes involved in rice Pi5-mediated disease resistance to Magnaporthe oryzae, we compared the proteomes of the RIL260 rice strain carrying the Pi5 resistance gene with its susceptible mutants M5465 and M7023. Proteins were extracted from the leaf tissues of both RIL260 and the mutant lines at 0, 24, and 48 h after M. oryzae inoculation and separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified eight proteins that were differently expressed between the resistant and susceptible plants (three down- and five up-regulated proteins in the mutants). The down-regulated proteins included a triosephosphate isomerase (spot no. 2210), a 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (no. 3611), and an unknown protein (no. 4505). In addition, the five up-regulated proteins in the mutants were predicted to be a fructokinase I (no. 313), a glutathione S-transferase (no. 2310), an atpB of chloroplast ATP synthase (no. 3616), an aminopeptidase N (no. 3724), and an unknown protein (no. 308). These results suggest that proteomic analysis of rice susceptible mutants is a useful method for identifying novel proteins involved in resistance to the M. oryzae pathogen.     

CRISPR/Cas9 in rice can induce new mutations in later generations,leading to chimerism and unpredicted segregation of the targeted mutation

CRISPR/Cas9 is a novel tool for targeted mutagenesis and is applicable to plants, including rice. Previous reports on CRISPR/Cas9 in rice have demonstrated that target mutations are transmitted to the next generation in accordance with Mendelian law, but heritability of the target mutation and the role of inherited Cas9 gene have not been fully elucidated. Here, we targeted the rice phytoene desaturase gene, mutants of which exhibit an albino phenotype, by using CRISPR/Cas9 and analyzed segregation of target mutations. Agrobacterium-mediated methods using immature embryos successfully transformed a CRISPR/Cas9 system into five rice cultivars and subsequently induced mutation. Unpredicted segregations, with more mutants than theoretically predicted, were frequently found in T₁ plants from monoallelic T₀ mutants. Chimeric plants with both biallelic and monoallelic mutated cells were also observed in the T₁. Next, we followed segregation of a target mutation in the T₂ from monoallelic T₁ mutants. When T₁ mutants possessed Cas9, unpredicted segregations of the target mutation and chimeric plants were observed again in the T₂. When T₁ mutants did not possess Cas9, segregation of the target mutations followed Mendelian law and no chimeric plants appeared in the T₂. T₂ mutants with Cas9 had mutations different from the original ones found in T₀. Our results indicated that inherited Cas9 was still active in later generations and could induce new mutations in the progeny, leading to chimerism and unpredicted segregation. We conclude that Cas9 has to be eliminated by segregation in T₁ to generate homozygous mutants without chimerism or unpredicted segregation.     

Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism

Key message

A method based on DNA single-strand conformation polymorphism is demonstrated for effective genotyping of CRISPR/Cas9-induced mutants in rice.

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has been widely adopted for genome editing in many organisms. A large proportion of mutations generated by CRISPR/Cas9 are very small insertions and deletions (indels), presumably because Cas9 generates blunt-ended double-strand breaks which are subsequently repaired without extensive end-processing. CRISPR/Cas9 is highly effective for targeted mutagenesis in the important crop, rice. For example, homozygous mutant seedlings are commonly recovered from CRISPR/Cas9-treated calli. However, many current mutation detection methods are not very suitable for screening homozygous mutants that typically carry small indels. In this study, we tested a mutation detection method based on single-strand conformational polymorphism (SSCP). We found it can effectively detect small indels in pilot experiments. By applying the SSCP method for CRISRP-Cas9-mediated targeted mutagenesis in rice, we successfully identified multiple mutants of OsROC5 and OsDEP1. In conclusion, the SSCP analysis will be a useful genotyping method for rapid identification of CRISPR/Cas9-induced mutants, including the most desirable homozygous mutants. The method also has high potential for similar applications in other plant species.

     

Efficient genetic transformation and CRISPR/Cas9‐mediated genome editing in Lemna aequinoctialis

The fast growth, ease of metabolic labelling and potential for feedstock and biofuels production make duckweeds not only an attractive model system for understanding plant biology, but also a potential future crop. However, current duckweed research is constrained by the lack of efficient genetic manipulation tools. Here, we report a case study on genome editing in a duckweed species, Lemna aequinoctialis, using a fast and efficient transformation and CRISPR/Cas9 tool. By optimizing currently available transformation protocols, we reduced the duration time of Agrobacterium‐mediated transformation to 5–6 weeks with a success rate of over 94%. Based on the optimized transformation protocol, we generated 15 (14.3% success rate) biallelic LaPDS mutants that showed albino phenotype using a CRISPR/Cas9 system. Investigations on CRISPR/Cas9‐mediated mutation spectrum among mutated L. aequinoctialis showed that most of mutations were short insertions and deletions. This study presents the first example of CRISPR/Cas9‐mediated genome editing in duckweeds, which will open new research avenues in using duckweeds for both basic and applied research.