Magnaporthe Oryzae Chloroplast-Targeting Endo-β-1,4-Xylanase I MoXYL1A Regulates Conidiation, Appressorium Maturation and Virulence of the Rice Blast Fungus

Endo-β-1,4-Xylanases are a group of extracellular enzymes that catalyze the hydrolysis of xylan, a principal constituent of the plant primary cell wall. The contribution of Endo-β-1,4-Xylanase I to both physiology and pathogenesis of the rice blast fungus M. oryzae is unknown. Here, we characterized the biological function of two endoxylanase I (MoXYL1A and MoXYL1B) genes in the development of M. oryzae using targeted gene deletion, biochemical analysis, and fluorescence microscopy. Phenotypic analysis of ∆Moxyl1A strains showed that MoXYL1A is required for the full virulence of M. oryzae but is dispensable for the vegetative growth of the rice blast fungus. MoXYL1B, in contrast, did not have a clear role in the infectious cycle but has a critical function in asexual reproduction of the fungus. The double deletion mutant was severely impaired in pathogenicity and virulence as well as asexual development. We found that MoXYL1A deletion compromised appressorium morphogenesis and function, leading to failure to penetrate host cells. Fluorescently tagged MoXYL1A and MoXYL1B displayed cytoplasmic localization in M. oryzae, while analysis of MoXYL1A-GFP and MoXYL1B-GFP in-planta revealed translocation and accumulation of these effector proteins into host cells. Meanwhile, sequence feature analysis showed that MoXYL1A possesses a transient chloroplast targeting signal peptide, and results from an Agrobacterium infiltration assay confirmed co-localization of MoXYL1A-GFP with ChCPN10C-RFP in the chloroplasts of host cells. MoXYL1B, accumulated to the cytoplasm of the host. Taken together, we conclude that MoXYL1A is a secreted effector protein that likely promotes the virulence of M. oryzae by interfering in the proper functioning of the host chloroplast, while the related xylanase MoXYL1B does not have a major role in virulence of M. oryzae.

Retracing the evolution from antimicrobial to targeting peptides

We experimentally challenged the endosymbiotic hypothesis that organelle-targeting peptides derive from antimicrobial amphipathic peptides delivered by the host cell, to which organelle progenitors became resistant. To explore the molecular changes required to convert such antimicrobial peptides into bona fide organelle-targeting peptides, we expressed a set of 13 antimicrobial peptides of various origins in the green alga Chlamydomonas reinhardtii that serves as a model for both mitochondrial and chloroplast import. The peptides were modified to match distinctive features of mitochondrial and chloroplast targeting peptides, and we assessed their targeting potential by following the intracellular localization and maturation of a Venus fluorescent reporter used as cargo protein. We present a temporal evolutionary scenario that emphasizes the early contribution of exchanging Lysines with Arginines in the sequence of the antimicrobial peptide, the evolution of a processing site followed by the addition of unstructured sequence and protein interaction sites that allow the selective targeting to the chloroplast.

Chloroplast transit peptides often require downstream unstructured sequence in Chlamydomonas reinhardtii

The N-terminal sequence stretch that defines subcellular targeting for most nuclear encoded chloroplast proteins is usually considered identical to the sequence that is cleaved upon import. Yet here this study shows that for nine out of ten tested Chlamydomonas chloroplast transit peptides, additional sequence past the cleavage site is required to enable chloroplast targeting. Using replacements of native post-cleavage residues with alternative sequences points to a role for unstructured sequence at mature protein N-termini.

Identification, Gene Structure, and Expression of BnMicEmUP: A Gene Upregulated in Embryogenic Brassica napus Microspores

Microspores of Brassica napus can be diverted from normal pollen development into embryogenesis by treating them with a mild heat shock. As microspore embryogenesis closely resembles zygotic embryogenesis, it is used as model for studying the molecular mechanisms controlling embryo formation. A previous study comparing the transcriptomes of three-day-old sorted embryogenic and pollen-like (non-embryogenic) microspores identified a gene homologous to AT1G74730 of unknown function that was upregulated 8-fold in the embryogenic cells. In the current study, the gene was isolated and sequenced from B. napus and named BnMicEmUP (B. napus microspore embryogenesis upregulated gene). Four forms of BnMicEmUP mRNA and three forms of genomic DNA were identified. BnMicEmUP2,3 was upregulated more than 7-fold by day 3 in embryogenic microspore cultures compared to non-induced cultures. BnMicEmUP1,4 was highly expressed in leaves. Transient expression studies of BnMicEmUP3::GFP fusion protein in Nicotiana benthamiana and in stable Arabidopsis transgenics showed that it accumulates in chloroplasts. The features of the BnMicEmUP protein, which include a chloroplast targeting region, a basic region, and a large region containing 11 complete leucine-rich repeats, suggest that it is similar to a bZIP PEND (plastid envelope DNA-binding protein) protein, a DNA binding protein found in the inner envelope membrane of developing chloroplasts. Here, we report that the BnMicEmUP3 overexpression in Arabidopsis increases the sensitivity of seedlings to exogenous abscisic acid (ABA). The BnMicEmUP proteins appear to be transcription factors that are localized in plastids and are involved in plant responses to biotic and abiotic environmental stresses; as well as the results obtained from this study can be used to improve crop yield.

Engineering 6-phosphogluconate dehydrogenase improves grain yield in heat-stressed maize

Endosperm starch synthesis is a primary determinant of grain yield and is sensitive to high-temperature stress. The maize chloroplast-localized 6-phosphogluconate dehydrogenase (6PGDH), PGD3, is critical for endosperm starch accumulation. Maize also has two cytosolic isozymes, PGD1 and PGD2, that are not required for kernel development. We found that cytosolic PGD1 and PGD2 isozymes have heat-stable activity, while amyloplast-localized PGD3 activity is labile under heat stress conditions. We targeted heat-stable 6PGDH to endosperm amyloplasts by fusing the Waxy1 chloroplast targeting the peptide coding sequence to the Pgd1 and Pgd2 open reading frames (ORFs). These WPGD1 and WPGD2 fusion proteins import into isolated chloroplasts, demonstrating a functional targeting sequence. Transgenic maize plants expressing WPGD1 and WPGD2 with an endosperm-specific promoter increased 6PGDH activity with enhanced heat stability in vitro. WPGD1 and WPGD2 transgenes complement the pgd3-defective kernel phenotype, indicating the fusion proteins are targeted to the amyloplast. In the field, the WPGD1 and WPGD2 transgenes can mitigate grain yield losses in high–nighttime-temperature conditions by increasing kernel number. These results provide insight into the subcellular distribution of metabolic activities in the endosperm and suggest the amyloplast pentose phosphate pathway is a heat-sensitive step in maize kernel metabolism that contributes to yield loss during heat stress.

Identification and expression analysis of chloroplast ribonucleoproteins (cpRNPs) in Arabidopsis and rice

Chloroplast ribonucleoproteins (cpRNPs) are implicated in splicing, editing and stability control of chloroplast RNAs as well as in regulating development and stress tolerance. To facilitate a comprehensive understanding of their functions, we carried out a genome-wide identification, curation, and phylogenetic analysis of cpRNP genes in Oryza sativa (rice) and Arabidopsis thaliana (Arabidopsis). Ten cpRNP genes were identified in each of Arabidopsis and rice genomes based on the presence of two RRM (RNA recognition motif) domains and an N-terminal chloroplast targeting signal peptide in the predicted proteins. These proteins are localized to chloroplasts. Gene expression analysis revealed that cpRNPs have differential tissue expression patterns and some cpRNPs are induced by abiotic stresses such as cold, heat and drought. Taken together, our study provides a comprehensive annotation of the cpRNP gene family and their expression patterns in Arabidopsis and rice which will facilitate further studies on their roles in plant growth and stress responses.

Characterization of PtAOS1 Promoter and Three Novel Interacting Proteins Responding to Drought in Poncirus trifoliata

Jasmonic acid (JA) plays a crucial role in various biological processes including development, signal transduction and stress response. Allene oxide synthase (AOS) catalyzing (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT) to an unstable allene oxide is involved in the first step of JA biosynthesis. Here, we isolated the PtAOS1 gene and its promoter from trifoliate orange (Poncirus trifoliata). PtAOS1 contains a putative chloroplast targeting sequence in N-terminal and shows relative to pistachio (Pistacia vera) AOS. A number of stress-, light- and hormone-related cis-elements were found in the PtAOS1 promoter which may be responsible for the up-regulation of PtAOS1 under drought and JA treatments. Transient expression in tobacco (Nicotiana benthamiana) demonstrated that the P−532 (−532 to +1) fragment conferring drive activity was a core region in the PtAOS1 promoter. Using yeast one-hybrid, three novel proteins, PtDUF886, PtDUF1685 and PtRAP2.4, binding to P−532 were identified. The dual luciferase assay in tobacco illustrated that all three transcription factors could enhance PtAOS1 promoter activity. Genes PtDUF1685 and PtRAP2.4 shared an expression pattern which was induced significantly by drought stress. These findings should be available evidence for trifoliate orange responding to drought through JA modulation.

Overexpression of Thalassiosira pseudonana violaxanthin de-epoxidase-like 2 (VDL2) increases fucoxanthin while stoichiometrically reducing diadinoxanthin cycle pigment abundance

ABSTRACTDespite the ubiquity and ecological importance of diatoms, much remains to be understood about their physiology and metabolism, including their carotenoid biosynthesis pathway. Early carotenoid biosynthesis steps are well-conserved, while the identity of the enzymes that catalyze the later steps and their order remain unclear. Those steps lead to the biosynthesis of the final pathway products: the main accessory light-harvesting pigment fucoxanthin (Fx) and the main photoprotective pigment pool comprised of diadinoxanthin (Ddx) and its reversibly de-epoxidized form diatoxanthin (Dtx). We used sequence comparison to known carotenoid biosynthesis enzymes to identify novel candidates in the diatom Thalassiosira pseudonana. Microarray and RNA-seq data was used to select candidates with transcriptomic responses similar to known carotenoid biosynthesis genes and to create full-length gene models, and we focused on those that encode proteins predicted to be chloroplast-localized. We identified a violaxanthin de-epoxidase-like gene (Thaps3_11707, VDL2) that when overexpressed results in increased Fx abundance while stoichiometrically reducing Ddx+Dtx. Based on transcriptomics, we hypothesize that Thaps3_10233 may also contribute to Fx biosynthesis, in addition to VDL2. Separately using antisense RNA to target VDL2, VDL1, and both LUT1-like copies (hypothesized to catalyze an earlier step in the pathway) simultaneously, reduced the overall cellular photosynthetic pigment content, including chlorophylls, suggesting destabilization of light-harvesting complexes by Fx deficiency. Based on transcriptomic and physiological data, we hypothesize that the two predicted T. pseudonana zeaxanthin epoxidases have distinct functions and that different copies of phytoene synthase and phytoene desaturase may serve to initiate carotenoid biosynthesis in response to different cellular needs. Finally, nine carotene cis/trans isomerase (CRTISO) candidates identified based on sequence identity to known CRTISO proteins were narrowed to two most likely to be part of the T. pseudonana carotenoid biosynthesis pathway based on transcriptomic responses and predicted chloroplast targeting.