Autophagy Benefits the Replication of Newcastle Disease Virus in Chicken Cells and Tissues

Newcastle disease virus (NDV) is an important avian pathogen. We previously reported that NDV triggers autophagy in U251 glioma cells, resulting in enhanced virus replication. In this study, we investigated whether NDV triggers autophagy in chicken cells and tissues to enhance virus replication. We demonstrated that NDV infection induced steady-state autophagy in chicken-derived DF-1 cells and in primary chicken embryo fibroblast (CEF) cells, evident through increased double- or single-membrane vesicles, the accumulation of green fluorescent protein (GFP)-LC3 dots, and the conversion of LC3-I to LC3-II. In addition, we measured autophagic flux by monitoring p62/SQSTM1 degradation, LC3-II turnover, and GFP-LC3 lysosomal delivery and proteolysis, to confirm that NDV infection induced the complete autophagic process. Inhibition of autophagy by pharmacological inhibitors and RNA interference reduced virus replication, indicating an important role for autophagy in NDV infection. Furthermore, we conducted in vivo experiments and observed the conversion of LC3-I to LC3-II in heart, liver, spleen, lung, and kidney of NDV-infected chickens. Regulation of the induction of autophagy with wortmannin, chloroquine, or starvation treatment affects NDV production and pathogenesis in tissues of both lung and intestine; however, treatment with rapamycin, an autophagy inducer of mammalian cells, showed no detectable changes in chicken cells and tissues. Moreover, administration of the autophagy inhibitor wortmannin increased the survival rate of NDV-infected chickens. Our studies provide strong evidence that NDV infection induces autophagy which benefits NDV replication in chicken cells and tissues.     

<Emphasis Type="Italic">qNCLB7.02</Emphasis>, a novel QTL for resistance to northern corn leaf blight in maize

Northern corn leaf blight (NCLB), which is caused by the hemibiotrophic fungal pathogen Setosphaeria turcica, is a devastating foliar disease that results in considerable maize yield losses. In the present study, quantitative trait locus (QTL) analysis was conducted across two environments using an ultra-high-density bin map constructed using recombinant inbred lines (RILs) derived from a cross between Ye478 and Qi319. A total of 11 QTLs, located on chromosomes 1, 4, 5, 6, 7, 8, 9, and 10, were detected that confer resistance to physiological race 0 of NCLB. Each QTL could explain 3.53–15.29% of the total phenotypic variation in disease resistance after artificial inoculation in two environments. Among these QTL, qNCLB7.02, which is located on chromosome 7, had the largest effect, accounting for 10.11 and 15.29% of the phenotypic variation in resistance in two field trials and BLUP. The common confidence interval (CI) for qNCLB7.02 was 1.4 Mb, according to the B73 RefGen_v3 sequence. The resistance effect of qNCLB7.02 was validated in 2016 by using chromosome segment substitution lines (CSSLs) derived from Qi319 as the donor in the genetic background of Ye478. The type 6 CSSL, which harbors introgressed qNCLB7.02, was found to be significantly associated with resistance to NCLB by linked marker bnlg1808 and exhibited greater resistance than the other CSSLs that did not carry this QTL (P?=?0.0008). The combination of linkage mapping in RILs and validation in CSSLs is a powerful approach for the dissection of QTL for disease resistance in maize.     

<Emphasis Type="Italic">Pm61</Emphasis>: a recessive gene for resistance to powdery mildew in wheat landrace Xuxusanyuehuang identified by comparative genomics analysis

Key message

A single recessive powdery mildew resistance gene Pm61 from wheat landrace Xuxusanyuehuang was mapped within a 0.46-cM genetic interval spanning a 1.3-Mb interval of the genomic region of chromosome arm 4AL.

Abstract

Epidemics of powdery mildew incited by the biotrophic fungus Blumeria graminis f. sp. tritici (Bgt) have caused significant yield reductions in many wheat (Triticum aestivum)-producing regions. Identification of powdery mildew resistance genes is required for sustainable improvement of wheat for disease resistance. Chinese wheat landrace Xuxusanyuehuang was resistant to several Bgt isolates at the seedling stage. Genetic analysis based on the inoculation of Bgt isolate E09 on the F₁, F₂, and F_2:3 populations produced by crossing Xuxusanyuehuang to susceptible cultivar Mingxian 169 revealed that the resistance of Xuxusanyuehuang was controlled by a single recessive gene. Bulked segregant analysis and simple sequence repeat (SSR) mapping placed the gene on chromosome bin 4AL-4-0.80-1.00. Comparative genomics analysis was performed to detect the collinear genomic regions of Brachypodium distachyon, rice, sorghum, Aegilops tauschii, T. urartu, and T. turgidum ssp. dicoccoides. Based on the use of 454 contig sequences and the International Wheat Genome Sequence Consortium survey sequence of Chinese Spring wheat, four EST-SSR and seven SSR markers were linked to the gene. An F₅ recombinant inbred line population derived from Xuxusanyuehuang?×?Mingxian 169 cross was used to develop the genetic linkage map. The gene was localized in a 0.46-cM genetic interval between Xgwm160 and Xicsx79 corresponding to 1.3-Mb interval of the genomic region in wheat genome. This is a new locus for powdery mildew resistance on chromosome arm 4AL and is designated Pm61.

     

极小种群野生植物对开蕨种群结构特征和群落物种多样性

以国家Ⅱ级保护极小种群野生植物——对开蕨(Phyllitis scolopendrium)为研究对象,分析了在海拔729、1008m群落内,其种群大小、分布频度和密度,个体形态特征指标及其在种群内、种群间差异,苗高分布规律、相对苗高组替代年龄级结构,分布格局和群落各层次的物种多样性,群落的相似性。结果表明:对开蕨在自然分布区内为偶见种,呈斑块状分布。在400m~2内,海拔1008m处(01群落)种群密度为31株,样地分布频度为43.75%,最大密度15株/25m~2;海拔729m处(02群落)种群密度为91株,样地分布频度为93.75%,最大密度30株/25m~2。通过对其自然苗高,叶片数量,有孢子囊叶片数量,最大叶片长、宽值,最小叶片长、宽值,冠径,叶片厚度7个形态指标的分析显示,种群内变异较大,随着植株高度(年龄)的增加,其变异系数均随之减小而趋于稳定;种群间在自然苗高,最大叶片长度和宽度,成熟孢子叶片数量,冠径,叶片厚度上达到极显著差异(P0.01)。其苗高分布呈现双峰型,01种群波谷出现在15.1—20.0cm处;02种群波谷出现在10.1—15.0cm处,两群落均显示有一次较大的更新过程,同时01种群在40.1—45.0cm出现间断。年龄级分析得出,01种群划分为5个龄级,近于正态分布;02种群划分为4个龄级,为倒J型分布;两种群分别处于中龄期和幼龄期,没有出现衰退型年龄结构。格局分析得出,对开蕨种群均为聚集分布。两群落的乔木、灌木、草本层的Shannon-Wiener指数、Pielou均匀度指数、Marglef丰富度指数、生态优势度和种间相遇机率较低(与地带顶级植被相比)而且分布不均;对开蕨在两群落的草本层中重要值较低,仅为伴生种。两群落相似性分析显示,乔木、灌木、草本层的相似度指数分别为66.67%、69.23%和38.46%,草本层差异较大。     

具尾蓝隐藻(CHROOMONAS CAUDATA GEITLER)的研究 Ⅰ形态特征──光镜及扫描电镜观察

对具尾蓝隐藻进行了光学显微镜及扫描电镜的观察。扫描电镜观察证明具尾蓝隐藻具有明显的纵沟,细胞顶端具圆形凹口,表明该种有“口沟”存在。从而澄清了文献上对该种有无“口沟”相互矛盾或模糊不清的描述。     

螺旋藻培养液吸收CO2特性的研究

培养液中含有高浓度的ＨＣＯ和ＣＯ,活跃进行的ＣＯ２,ＨＣＯ和ＣＯ３种碳源形式相互转变的化学反应,构成了螺旋藻培养液不同于其它藻类培养液的显著特征。定量研究了ＣＯ２吸收速率与碳源浓度、温度、ｐＨ值、盐度、培养液运动状态的关系,利用培养液吸收ＣＯ２的物理模型解释了碳源浓度、ｐＨ值、培养液运动状态影响ＣＯ２吸收速率的机理。对化学反应是否影响ＣＯ２吸收速率这一有争议的问题进行了探讨,在肯定化学反应影响的前提下,指出化学反应的影响能否被观察到,显著程度如何,关键在于培养液的运动状态。根据实验结果,给出了利用“气罩法”添加ＣＯ２,所需气罩面积与产量、碳源浓度、培养液运动状态、培养面积数量关系的理论值。使用培养液的“ＣＯ２容量”的概念,说明利用ＣＯ２为碳源培养螺旋藻与其它藻类相比,可以得到更高的碳源利用率,从而产生更大的效益。     

The Exonuclease Homolog OsRAD1 Promotes Accurate Meiotic Double-Strand Break Repair by Suppressing Nonhomologous End Joining

During meiosis, programmed double-strand breaks (DSBs) are generated to initiate homologous recombination, which is crucial for faithful chromosome segregation. In yeast, Radiation sensitive1 (RAD1) acts together with Radiation sensitive9 (RAD9) and Hydroxyurea sensitive1 (HUS1) to facilitate meiotic recombination via cell-cycle checkpoint control. However, little is known about the meiotic functions of these proteins in higher eukaryotes. Here, we characterized a RAD1 homolog in rice (Oryza sativa) and obtained evidence that O. sativa RAD1 (OsRAD1) is important for meiotic DSB repair. Loss of OsRAD1 led to abnormal chromosome association and fragmentation upon completion of homologous pairing and synapsis. These aberrant chromosome associations were independent of OsDMC1. We found that classical nonhomologous end-joining mediated by Ku70 accounted for most of the ectopic associations in Osrad1. In addition, OsRAD1 interacts directly with OsHUS1 and OsRAD9, suggesting that these proteins act as a complex to promote DSB repair during rice meiosis. Together, these findings suggest that the 9-1-1 complex facilitates accurate meiotic recombination by suppressing nonhomologous end-joining during meiosis in rice.Meiosis comprises two successive cell divisions after a single S phase, generating four haploid products. To ensure proper chromosome segregation at the first meiotic division, crossovers (COs) are formed between homologous chromosomes (Kleckner, 2006). CO formation requires faithful repair of programmed DNA double-strand breaks (DSBs) introduced by the protein SPO11 (Keeney et al., 1997; Shinohara et al., 1997).Mitotic cells employ two basic strategies for DSB repair: homologous recombination (HR) and classical nonhomologous end-joining (C-NHEJ; Deriano and Roth, 2013). HR requires an undamaged template sequence for repair, while the C-NHEJ pathway involves direct ligation of the broken ends in a Ku-dependent manner. Both HR and C-NHEJ safeguard genome integrity during mitosis (Ceccaldi et al., 2016; Symington and Gautier, 2011). However, DSBs are preferentially repaired by HR during meiosis, because only this pathway generates COs. C-NHEJ competes with HR and creates de novo mutations in the gametes, indicating that this activity should be restricted during meiotic DSB repair. Previous studies have identified several factors essential for preventing C-NHEJ in meiosis (Goedecke et al., 1999; Martin et al., 2005; Adamo et al., 2010; Lemmens et al., 2013).Although the mechanism inhibiting C-NHEJ during meiosis is still elusive, regulators guaranteeing the success of the HR pathway have been extensively studied. Radiation sensitive1 (RAD1) is an evolutionarily conserved protein whose best-known function is checkpoint signaling. RAD1, a member of the ring-shaped RAD9-RAD1-HUS1 (9-1-1) complex, plays a crucial role in activating the pachytene checkpoint, a surveillance mechanism for monitoring the progression of meiotic HR in many organisms (Lydall et al., 1996; Hong and Roeder, 2002; Eichinger and Jentsch, 2010).In addition to their well-known roles in checkpoint signaling, members of 9-1-1 complex may also play a direct role in facilitating DSB repair and HR during meiosis. RAD1 is associated with both synapsed and unsynapsed chromosomes during prophase I in mouse (Freire et al., 1998). The homolog of RAD1 in Saccharomyces cerevisiae is Rad17, and rad17 mutant exhibits persistent Rad51 foci (Shinohara et al., 2003). Moreover, mutations in Rad17 lead to a reduced frequency of interhomolog recombination, aberrant synapsis, increased rates of ectopic recombination events, and illegitimate repair from the sister chromatids during meiosis (Grushcow et al., 1999). Recently, Rad17 was shown to be necessary for the efficient assembly of ZMM proteins (Shinohara et al., 2015). Apart from RAD1, the other partners of 9-1-1 were also shown to be involved in DSB repair. HUS1 is proved essential for meiotic DSB repair in Drosophila (Peretz et al., 2009). Moreover, Hus1 inactivation in mouse testicular germ cells results in persistent meiotic DNA damage, chromosomal defects, and germ cell depletion (Lyndaker et al., 2013). Nevertheless, little is known about the role of 9-1-1 proteins in higher plants. In Arabidopsis (Arabidopsis thaliana), mutants of RAD9 show increased sensitivity to genotoxic agents and delayed general repair of mitotic DSBs (Heitzeberg et al., 2004). A recent study indicates that HUS1 is involved in DSB repair of both mitotic and meiotic cells in rice (Che et al., 2014).In this study, we showed that OsRAD1 was required for the accurate repair of DSBs in rice during meiosis. Importantly, we demonstrated that the defective meiotic DSB repair in the Osrad1 mutants could be partially suppressed by blocking the C-NHEJ pathway. We also investigated the relationship between OsRAD1 and other key recombination proteins. Together, our findings indicated that the 9-1-1 complex plays a crucial role in the meiotic DSB repair mechanism.