Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA. Consistent with this finding, no off-target mutations are detected in the mutant plants. Because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing, the mutants obtained are completely transgene free. This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized.

Form I Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), a complex of eight large (RbcL) and eight small (RbcS) subunits, catalyses the fixation of atmospheric CO(2) in photosynthesis. The limited catalytic efficiency of Rubisco has sparked extensive efforts to re-engineer the enzyme with the goal of enhancing agricultural productivity. To facilitate such efforts we analysed the formation of cyanobacterial form I Rubisco by in vitro reconstitution and cryo-electron microscopy. We show that RbcL subunit folding by the GroEL/GroES chaperonin is tightly coupled with assembly mediated by the chaperone RbcX(2). RbcL monomers remain partially unstable and retain high affinity for GroEL until captured by RbcX(2). As revealed by the structure of a RbcL(8)-(RbcX(2))(8) assembly intermediate, RbcX(2) acts as a molecular staple in stabilizing the RbcL subunits as dimers and facilitates RbcL(8) core assembly. Finally, addition of RbcS results in RbcX(2) release and holoenzyme formation. Specific assembly chaperones may be required more generally in the formation of complex oligomeric structures when folding is closely coupled to assembly.

SummaryThe vesicle-inducing protein in plastids (VIPP1) is essential for the biogenesis of thylakoid membranes in cyanobacteria and plants. VIPP1 and its bacterial ancestor PspA form large homo-oligomeric rings of >1 MDa. We recently demonstrated that VIPP1 interacts with the chloroplast J-domain co-chaperone CDJ2 and its chaperone partner HSP70B, and hypothesized that the chaperones might be involved in the assembly and/or disassembly of VIPP1 oligomers. To test this hypothesis, we analysed the composition of VIPP1/chaperone complexes in Chlamydomonas reinhardtii cell extracts and monitored effects of the chaperones on VIPP1 assembly states in vitro. We found that CGE1, the chloroplast GrpE homologue, is also part of complexes with HSP70B, CDJ2 and VIPP1. We observed that CDJ2-VIPP1 accumulated as low-and high-molecular-weight complexes in ATP-depleted cell extracts, but as intermediate-size complexes in extracts supplemented with ATP. This was consistent with a role for the chaperones in VIPP1 assembly and disassembly. Using purified proteins, we could demonstrate that the chaperones indeed facilitated both the assembly and disassembly of VIPP1 oligomers. Electron microscopy studies revealed that, in contrast to PspA, VIPP1 rings assembled into rod-shaped supercomplexes that were morphologically similar to microtubule-like structures observed earlier in various plastid types. VIPP1 rods, too, were disassembled by the chaperones, and chaperone-mediated rod disassembly also occurred when VIPP1 lacked an approximately 30-aa C-terminal extension present in VIPP1 homologues but absent in PspA. By regulating the assembly state of VIPP1, the chloroplast HSP70 chaperone system may play an important role in the maintenance/biogenesis of thylakoid membranes.

J-domain cochaperones confer functional specificity to their heat shock protein (HSP)70 partner by recruiting it to specific substrate proteins. To gain insight into the functions of plastidic HSP70s, we searched in Chlamydomonas databases for expressed sequence tags that potentially encode chloroplast-targeted J-domain cochaperones. Two such cDNAs were found: the encoded J-domain proteins were named chloroplast DnaJ homolog 1 and 2 (CDJ1 and CDJ2). CDJ2 was shown to interact with a ϳ28-kDa protein that by mass spectrometry was identified as the vesicle-inducing protein in plastids 1 (VIPP1). In fractionation experiments, CDJ2 was detected almost exclusively in the stroma, whereas VIPP1 was found in low-density membranes, thylakoids, and in the stroma. Coimmunoprecipitation and mass spectrometry analyses identified stromal HSP70B as the major protein interacting with soluble VIPP1, and, as confirmed by cross-linking data, as chaperone partner of CDJ2. In blue native-PAGE of soluble cell extracts, CDJ2 and VIPP1 comigrated in complexes of Ͼ Ͼ669, ϳ150, and perhaps ϳ300 kDa. Our data suggest that CDJ2, presumably via coiled-coil interactions, binds to VIPP1 and presents it to HSP70B in the ATP state. Our findings and the previously reported requirement of VIPP1 for the biogenesis of thylakoid membranes point to a role for the HSP70B/CDJ2 chaperone pair in this process. INTRODUCTIONChaperones of the heat shock protein (Hsp)70 family belong to the most conserved proteins known. Except for some Archaea, Hsp70s are found in all known organisms and are present in every compartment of the eukaryotic cell (Bukau and Horwich, 1998). Principally, Hsp70s consist of an Nterminal ATPase domain and a C-terminal substrate-binding domain. ATP hydrolysis at the ATPase domain regulates substrate binding and release. Substrate proteins recognized by Hsp70 expose hydrophobic regions, a characteristic feature not only of nonnative proteins, but also of native Hsp70 substrates. Binding of Hsp70 to hydrophobic regions prevents the formation of aggregates. In addition, the intrinsic secondary amide peptide bond cis-trans isomerase activity recently detected for DnaK (the Hsp70 of Escherichia coli) may introduce conformational changes to bound substrates that eventually allow nonnative proteins to reconvert to the native state (Schiene-Fischer et al., 2002). Thus, Hsp70s play a major role in the folding of nascent chains and in the renaturation of nonnative proteins that have accumulated during stress situations such as heat shock (Frydman, 2001). However, they also are involved in many highly specialized functions such as the regulation of the general stress response (Tomoyasu et al., 1998), the uncoating of clathrincoated vesicles (Ungewickell et al., 1995), or the translocation of proteins across membranes (Kang et al., 1990).Specificity of Hsp70 function is mediated largely by its cochaperones, of which the J-domain cochaperones represent an important class. J-domain cochaperones contain a highly conserved J-domain that is responsibl...

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most destructive diseases that pose a great threat to wheat production. Wheat landraces represent a rich source of powdery mildew resistance. Here, we report the map-based cloning of powdery mildew resistance gene Pm24 from Chinese wheat landrace Hulutou. It encodes a tandem kinase protein (TKP) with putative kinase-pseudokinase domains, designated WHEAT TANDEM KINASE 3 (WTK3). The resistance function of Pm24 was validated by transgenic assay, independent mutants, and allelic association analyses. Haplotype analysis revealed that a rare 6-bp natural deletion of lysine-glycine codons, endemic to wheat landraces of Shaanxi Province, China, in the kinase I domain (Kin I) of WTK3 is critical for the resistance function. Transgenic assay of WTK3 chimeric variants revealed that only the specific two amino acid deletion, rather than any of the single or more amino acid deletions, in the Kin I of WTK3 is responsible for gaining the resistance function of WTK3 against the Bgt fungus.

SUMMARYPolyploidy is a common phenomenon, particularly in plants. The soybean (Glycine max [L.] Merr.) genome has undergone two whole genome duplication (WGD) events. The conservation and divergence of duplicated gene pairs are major contributors to genome evolution. D1 and D2 are two unlinked, paralogous nuclear genes, whose double-recessive mutant (d1d1d2d2) results in chlorophyll retention, called 'staygreen'. Through molecular cloning and functional analyses, we demonstrated that D1 and D2 are homologs of the STAY-GREEN (SGR) genes from other plant species and were duplicated as a result of the most recent WGD in soybean. Transcriptional analysis showed that both D1 and D2 were more highly expressed in older tissues, and chlorophyll degradation and programmed cell death-related genes were suppressed in a d1d2 double mutant, this situation indicated that these genes are probably involved in the early stages of tissue senescence. Investigation of genes that flank D1 and D2 revealed that evolution within collinear duplicated blocks may affect the conservation of individual gene pairs within the blocks. Moreover, we found that a long terminal repeat retrotransposon, GmD2IN, resulted in the d2 mutation. Further analysis of this retrotransposon family showed that insertion in or near the coding regions can affect gene expression or splicing patterns, and may be an important force to promote the divergence of duplicated gene pairs.

The individual roles of three chloroplast CPN60 protomers (CPN60α, CPN60β1, and CPN60β2) and whether and how they are assembled into functional chaperonin complexes are investigated in Chlamydomonas reinhardtii. Protein complexes containing all three potential subunits were identified in Chlamydomonas, and their co-expression in Escherichia coli yielded a homogeneous population of oligomers containing all three subunits (CPN60αβ1β2), with a molecular weight consistent with a tetradecameric structure. While homo-oligomers of CPN60β could form, they were dramatically reduced when CPN60α was present and homo-oligomers of CPN60β2 were readily changed into hetero-oligomers in the presence of ATP and other protomers. ATP hydrolysis caused CPN60 oligomers to disassemble and drove the purified protomers to reconstitute oligomers in vitro, suggesting that the dynamic nature of CPN60 oligomers is dependent on ATP. Only hetero-oligomeric CPN60αβ1β2, containing CPN60α, CPN60β1, and CPN60β2 subunits in a 5:6:3 ratio, cooperated functionally with GroES. The combination of CPN60α and CPN60β subunits, but not the individual subunits alone, complemented GroEL function in E. coli with subunit recognition specificity. Down-regulation of the CPN60α subunit in Chlamydomonas resulted in a slow growth defect and an inability to grow autotrophically, indicating the essential role of CPN60α in vivo.

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