The plant Pontin and Reptin homologues,RuvBL1 and RuvBL2a,colocalize with TERT and TRB proteins in vivo,and participate in telomerase biogenesis

Telomerase maturation and recruitment to telomeres is regulated by several telomerase‐ and telomere‐associated proteins. Among a number of proteins, human Pontin and Reptin play critical roles in telomerase biogenesis. Here we characterized plant orthologues of Pontin and Reptin, RuvBL1 and RuvBL2a, respectively, and show association of Arabidopsis thaliana RuvBL1 (AtRuvBL1) with the catalytic subunit of telomerase (AtTERT) in the nucleolus in vivo. In contrast to mammals, interactions between AtTERT and AtRuvBL proteins in A. thaliana are not direct and they are rather mediated by one of the Arabidopsis thaliana Telomere Repeat Binding (AtTRB) proteins. We further show that plant orthologue of dyskerin, named AtCBF5, is indirectly associated with AtTRB proteins but not with the AtRuvBL proteins in the plant nucleus/nucleolus, and interacts with the Protection of telomere 1 (AtPOT1a) in the nucleolus or cytoplasmic foci. Our genome‐wide phylogenetic analyses identify orthologues in RuvBL protein family within the plant kingdom. Dysfunction of AtRuvBL genes in heterozygous T‐DNA insertion A. thaliana mutants results in reduced telomerase activity and indicate the involvement of AtRuvBL in plant telomerase biogenesis.     

Dyskerin is a component of the Arabidopsis telomerase RNP required for telomere maintenance

Dyskerin binds the H/ACA box of human telomerase RNA and is a core telomerase subunit required for RNP biogenesis and enzyme function in vivo. Missense mutations in dyskerin result in dyskeratosis congenita, a complex syndrome characterized by bone marrow failure, telomerase enzyme deficiency, and progressive telomere shortening. Here we demonstrate that dyskerin also contributes to telomere maintenance in Arabidopsis thaliana. We report that both AtNAP57, the Arabidopsis dyskerin homolog, and AtTERT, the telomerase catalytic subunit, accumulate in the plant nucleolus, and AtNAP57 associates with active telomerase RNP particles in an RNA-dependent manner. Furthermore, AtNAP57 interacts in vitro with AtPOT1a, a novel component of Arabidopsis telomerase. Although a null mutation in AtNAP57 is lethal, AtNAP57, like AtTERT, is not haploinsufficient for telomere maintenance in Arabidopsis. However, introduction of an AtNAP57 allele containing a T66A mutation decreased telomerase activity in vitro, disrupted telomere length regulation on individual chromosome ends in vivo, and established a new, shorter telomere length set point. These results imply that T66A NAP57 behaves as a dominant-negative inhibitor of telomerase. We conclude that dyskerin is a conserved component of the telomerase RNP complex in higher eukaryotes that is required for maximal enzyme activity in vivo.     

Telomere repeat binding proteins are functional components of Arabidopsis telomeres and interact with telomerase

Although telomere‐binding proteins constitute an essential part of telomeres, in vivo data indicating the existence of a structure similar to mammalian shelterin complex in plants are limited. Partial characterization of a number of candidate proteins has not identified true components of plant shelterin or elucidated their functional mechanisms. Telomere repeat binding (TRB) proteins from Arabidopsis thaliana bind plant telomeric repeats through a Myb domain of the telobox type in vitro, and have been shown to interact with POT1b (Protection of telomeres 1). Here we demonstrate co‐localization of TRB1 protein with telomeres in situ using fluorescence microscopy, as well as in vivo interaction using chromatin immunoprecipitation. Classification of the TRB1 protein as a component of plant telomeres is further confirmed by the observation of shortening of telomeres in knockout mutants of the trb1 gene. Moreover, TRB proteins physically interact with plant telomerase catalytic subunits. These findings integrate TRB proteins into the telomeric interactome of A. thaliana.     

The spatiotemporal control of KatG2 catalase-peroxidase contributes to the invasiveness of Fusarium graminearum in host plants

Reactive oxygen species (ROS) are involved in the pathogen-host interactions, and play a Janus-faced role in the resistance and susceptibility of plants to biotrophic and necrotrophic pathogens. The ascomycete fungus Fusarium graminearum causes hazardous wheat Fusarium head blight worldwide. Deletion of the putative secreted catalase-peroxidase gene in F. graminearum, KatG2, reduced the virulence in wheat spike infection. However, it remains unclear when and where KatG2 scavenges ROS during the invasion of wheat. In this study, we delineate the change in ROS levels in the transition of the infection phase under microscopic observation. Correspondingly, the pathogen switches its strategy of infection with temporal and spatial regulation of KatG2 to counteract oxidative stress generated by host plant cells. With the native promoter-driven KatG2-mRFP strain, we show that KatG2-mRFP expression was induced in planta and accumulated in the infection front region at the early infection stage. In contrast to its ubiquitous cellular localization in runner hyphae, KatG2-mRFP is exclusively located on the cell wall of invading hyphal cells, especially at the pathogen-host cellular interface. Using posttranslational modification analysis, we found that asparagine residues at the 238 and 391 positions of KatG2 could be modified by N-glycosylation and that these two residues are required for KatG2 accumulation and cell wall localization in planta.     

Sorting nexin Snx41 is essential for conidiation and mediates glutathione-based antioxidant defense during invasive growth in Magnaporthe oryzae

The sorting nexins Atg20/Snx42 and Snx41 regulate membrane traffic and endosomal protein sorting and are essential for Cvt and/or pexophagy in yeast. Previously, we showed that macroautophagy is necessary for conidiation in the rice-blast fungus Magnaporthe oryzae. Here, we analyzed the physiological function(s) of selective autophagy in Magnaporthe through targeted deletion of MGG_12832, an ortholog of yeast SNX41 and ATG20/SNX42. Loss of MGG_12832 (hereafter SNX41) abolished conidia formation and pathogenesis in M. oryzae. Snx41-GFP localized as dynamic puncta or short tubules that are partially associated with autophagosomes and/or autophagic vacuoles. PX domain, but not macroautophagy per se, was required for such localization of Snx41-GFP in Magnaporthe. Although not required for nonselective autophagy, Snx41 was essential for pexophagy in Magnaporthe. We identified Oxp1, an ATP-dependent oxoprolinase in the gamma-glutamyl cycle, as a binding partner and potential retrieval target of Snx41-dependent protein sorting. The substrate of Oxp1, 5-oxoproline, could partially restore conidiation in the snx41Δ. Exogenous glutathione, a product of the gamma-glutamyl cycle, significantly restored pathogenicity in the snx41Δ mutant, likely through counteracting the oxidative stress imposed by the host. We propose that the gamma-glutamyl cycle and glutathione biosynthesis are subject to regulation by Snx41-dependent vesicular trafficking, and mediate antioxidant defense crucial for in planta growth and pathogenic differentiation of Magnaporthe at the onset of blast disease in rice.     

The <Emphasis Type="Italic">Arabidopsis</Emphasis> LHP1 protein is a component of euchromatin

The HP1 family proteins are involved in several aspects of chromatin function and regulation in Drosophila, mammals and the fission yeast. Here we investigate the localization of LHP1, the unique Arabidopsis thaliana HP1 homolog known at present time, to approach its function. A functional LHP1–GFP fusion protein, able to restore the wild-type phenotype in the lhp1 mutant, was used to analyze the subnuclear distribution of LHP1 in both A. thaliana and Nicotiana tabacum. In A. thaliana interphase nuclei, LHP1 was predominantly located outside the heterochromatic chromocenters. No major aberrations were observed in heterochromatin content or chromocenter organization in lhp1 plants. These data indicate that LHP1 is mainly involved in euchromatin organization in A. thaliana. In tobacco BY-2 cells, the LHP1 distribution, although in foci, slightly differed suggesting that LHP1 localization is determined by the underlying genome organization of plant species. Truncated LHP1 proteins expressed in vivo allowed us to determine the function of the different segments in the localization. The in foci distribution is dependent on the presence of the two chromo domains, whereas the hinge region has some nucleolus-targeting properties. Furthermore, like the animal HP1β and HP1γ subtypes, LHP1 dissociates from chromosomes during mitosis. In transgenic plants expressing the LHP1–GFP fusion protein, two major localization patterns were observed according to cell types suggesting that localization evolves with age or differentiation states. Our results show conversed characteristics of the A. thaliana HP1 homolog with the mammal HP1γ isoform, besides specific plant properties.     

A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus

The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by the Madagascar periwinkle (Catharanthus roseus). Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003–0.01% yields. Metabolic engineering efforts to either improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. A VIGS method was developed herein to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro.     

New Insight into the Structure and Regulation of the Plant Vacuolar H+-ATPase

Plant cells are characterized by a highly active secretory system that includes the large central vacuole found in most differentiated tissues. The plant vacuolar H⁺-ATPase plays an essential role in maintaining the ionic and metabolic gradients across endomembranes, in activating transport processes and vesicle dynamics, and, hence, is indispensable for plant growth, development, and adaptation to changing environmental conditions. The review summarizes recent advances in elucidating the structure, subunit composition, localization, and regulation of plant V-ATPase. Emerging knowledge on subunit isogenes from Arabidopsis and rice genomic sequences as well as from Mesembryanthemum illustrates another level of complexity, the regulation of isogene expression and function of subunit isoforms. To this end, the review attempts to define directions of future research on plant V-ATPase.     

Detection of Telomerase Activity in Tissues and Primary Cultured Lymphoid Cells of <Emphasis Type="Italic">Penaeus japonicus</Emphasis>

Telomerase is a ribonucleoprotein enzyme that can elongate telomeric DNA, which is thought to be required for the development of cellular immortality and oncogenesis in mammals. We examined telomerase activity in tissues and primary cultured lymphoid cells of adult penaeid shrimps. Using the telomeric repeat amplification protocol (TRAP), we studied the characteristics of a putative novel telomerase in Penaeus japonicus. This telomerase could be inactivated by heating or treatment with RNase A or proteinase K. At elongation, this telomerase required dATP, dGTP, and dTTP, but not dCTP, as substrates. Sequence analysis of the TRAP product revealed that this telomerase synthesized (TTAGG)_n repeated sequences. The activity of this telomerase was decreased but still readily detectable in 100 ng of protein extract from lymphoid tissue. The telomerase activity was detected in all examined tissues including testis, ovary, lymphoid, heart, hepatopancreas, and muscle. The highest telomerase activity was in the extract of ovarian tissues. In primary cultured lymphoid cells, the telomerase activity was retained. Thus, primary cultured lymphoid cells of Penaeus japonicus possess one of the factors necessary for cell line establishment.