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      Identification of Autophagy in the Pine Wood Nematode Bursaphelenchus xylophilus and the Molecular Characterization and Functional Analysis of Two Novel Autophagy-Related Genes, BxATG1 and BxATG8

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          Abstract

          The pine wood nematode, Bursaphelenchus xylophilus, causes huge economic losses in pine forests, has a complex life cycle, and shows the remarkable ability to survive under unfavorable and changing environmental conditions. This ability may be related to autophagy, which is still poorly understood in B. xylophilus and no autophagy-related genes have been previously characterized. In this study, transmission electron microscopy was used to confirm that autophagy exists in B. xylophilus. The full-length cDNAs of BxATG1 and BxATG8 were first cloned from B. xylophilus, and BxATG1 and BxATG8 were characterized using bioinformatics methods. The expression pattern of the autophagy marker BxATG8 was investigated using in situ hybridization (ISH). BxATG8 was expressed in esophageal gland and hypodermal seam cells. We tested the effects of RNA interference (RNAi) on BxATG1 and BxATG8. The results revealed that BxATG1 and BxATG8 were likely associated with propagation of nematodes on fungal mats. This study confirmed the molecular characterization and functions of BxATG1 and BxATG8 in B. xylophilus and provided fundamental information between autophagy and B. xylophilus.

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          Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization.

          We sought to create a comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle. To this end, we used DNA microarrays and samples from yeast cultures synchronized by three independent methods: alpha factor arrest, elutriation, and arrest of a cdc15 temperature-sensitive mutant. Using periodicity and correlation algorithms, we identified 800 genes that meet an objective minimum criterion for cell cycle regulation. In separate experiments, designed to examine the effects of inducing either the G1 cyclin Cln3p or the B-type cyclin Clb2p, we found that the mRNA levels of more than half of these 800 genes respond to one or both of these cyclins. Furthermore, we analyzed our set of cell cycle-regulated genes for known and new promoter elements and show that several known elements (or variations thereof) contain information predictive of cell cycle regulation. A full description and complete data sets are available at http://cellcycle-www.stanford.edu
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            Construct design for efficient, effective and high-throughput gene silencing in plants.

            Post-transcriptional silencing of plant genes using anti-sense or co-suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti-sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron-containing constructs (ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co-suppression or anti-sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high-throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.
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              The molecular machinery of autophagy: unanswered questions.

              Autophagy is a process in which cytosol and organelles are sequestered within double-membrane vesicles that deliver the contents to the lysosome/vacuole for degradation and recycling of the resulting macromolecules. It plays an important role in the cellular response to stress, is involved in various developmental pathways and functions in tumor suppression, resistance to pathogens and extension of lifespan. Conversely, autophagy may be associated with certain myopathies and neurodegenerative conditions. Substantial progress has been made in identifying the proteins required for autophagy and in understanding its molecular basis; however, many questions remain. For example, Tor is one of the key regulatory proteins at the induction step that controls the function of a complex including Atg1 kinase, but the target of Atg1 is not known. Although autophagy is generally considered to be nonspecific, there are specific types of autophagy that utilize receptor and adaptor proteins such as Atg11; however, the means by which Atg11 connects the cargo with the sequestering vesicle, the autophagosome, is not understood. Formation of the autophagosome is a complex process and neither the mechanism of vesicle formation nor the donor membrane origin is known. The final breakdown of the sequestered cargo relies on well-characterized lysosomal/vacuolar proteases; the roles of lipases, by contrast, have not been elucidated, and we do not know how the integrity of the lysosome/vacuole membrane is maintained during degradation.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                03 March 2016
                March 2016
                : 17
                : 3
                : 279
                Affiliations
                [1 ]Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, Jiangsu, China; denglina121@ 123456163.com (L.-N.D.); jrye@ 123456njfu.edu.cn (J.-R.Y.); xq2159@ 123456163.com (Q.X.)
                [2 ]Jiangsu Key Laboratory for Prevention and Management of Invasive Species, Nanjing Forestry University, Nanjing 210037, Jiangsu, China
                [3 ]Yancheng Institute of Technology, School of Ocean and Biological Engineering, Yancheng 224051, Jiangsu, China
                Author notes
                [* ]Correspondence: xqwu@ 123456njfu.edu.cn ; Tel./Fax: +86-25-8542-7427
                Article
                ijms-17-00279
                10.3390/ijms17030279
                4813143
                26950119
                ce4b284d-5c9c-4cbe-af38-1c1a95a0bcee
                © 2016 by the authors; licensee MDPI, Basel, Switzerland.

                This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 09 January 2016
                : 14 February 2016
                Categories
                Article

                Molecular biology
                bursaphelenchus xylophilus,autophagy,transmission electron microscopy,autophagy-related genes,in situ hybridization,rna interference

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