The primary functions of mitochondria are to produce energy and participate in the apoptosis of cells, with them being highly conserved among eukaryotes. However, the composition of mitochondrial genomes, mitochondrial DNA (mtDNA) replication, and mitochondrial inheritance varies significantly among animals, plants, and fungi. Especially in fungi, there exists a rich diversity of mitochondrial genomes, as well as various replication and inheritance mechanisms. Therefore, a comprehensive understanding of fungal mitochondria is crucial for unraveling the evolutionary history of mitochondria in eukaryotes. In this review, we have organized existing reports to systematically describe and summarize the composition of yeast-like fungal mitochondrial genomes from three perspectives: mitochondrial genome structure, encoded genes, and mobile elements. We have also provided a systematic overview of the mechanisms in mtDNA replication and mitochondrial inheritance during bisexual mating. Additionally, we have discussed and proposed open questions that require further investigation for clarification.

Inflammatory responses and tumor developments are closely related, with interleukin-6 (IL-6) playing important roles in both processes. IL-6 has been extensively identified as a potential tumor biomarker. This study developed an isotope dilution mass spectrometry (IDMS) method for quantifying IL-6 based on signature peptides. These peptides were screened by excluding those with missed cleavage or post-translational modification. The method’s accuracy was verified using amino acid-based IDMS, in which purified IL-6 protein samples were quantified after hydrolyzing them into amino acids, and no significant difference was observed (p-value < 0.05). The method demonstrated good linearity and sensitivity upon testing. The specificity and matrix effect of the method were verified, and a precision study showed that the coefficient of variation was less than 5% for both the intra-day and inter-day tests. Compared to immunoassays, this method offers distinct advantages, such as the facilitation of multi-target analysis. Furthermore, the peptides used in this study are much more convenient for storage and operation than the antibodies or purified proteins typically used in immunoassays.

通过建立适用于菰黑粉菌Ustilago esculenta的农杆菌介导遗传转化(Agrobacterium tumefaciens-mediated transformation,ATMT)体系,构建菰黑粉菌T-DNA插入突变体库.针对性地筛选双核菌丝形成缺陷型转化子,并对T-DNA插入位点进行分析,为研究菰黑粉菌二态型转换的分子调控机理打下基础.以构建的菰黑粉菌自融合菌株TSP为出发菌株,以含有遗传霉素(G418)抗性基因(neo)的质粒为载体,通过ATMT构建菰黑粉菌T-DNA突变体库,并对诱导剂乙酰丁香酮(AS)浓度、转化的共培养时间、农杆菌浓度和菰黑粉菌芽孢子浓度等建库影响因素进行单因素条件试验,筛选最优条件;对继代培养的转化子基因组中的遗传霉素抗性基因进行PCR检测,验证转化子遗传稳定性;对突变体库中的转化子双核菌丝生长情况进行观察,测定其双核菌丝形成能力;对上述双核菌丝形成缺陷型转化子进行基因组重测序,分析其T-DNA插入位点.当遗传霉素浓度为75 μg/mL时,菰黑粉菌的生长被完全抑制.当AS浓度为100μg/mL、共培养时间为24 h、孢子浓度为1×105个/mL、农杆菌浓度为OD600=0.3时,转化获得转化子的效率最高,为菰黑粉菌ATMT最优转化体系.在突变体库中随机选取7株转化子在YEPS固体平板上继代培养10代,仍然能够通过PCR的方法在基因组中检测到neo基因片段,说明T-DNA成功插入TSP菌株基因组且稳定遗传.针对部分转化子进行双核菌丝生长能力测定,有5株转化子的菌落边缘没有形成菌丝,而TSP菌株的边缘长出了明显的菌丝,说明这5株转化子双核菌丝形成的能力丧失.对上述双核菌丝形成缺陷型转化子中的其中2个(TSP-1、TSP-23)进行基因组重测序,比对结果显示,TSP-1插入位点位于其交配型基因a位点的(GenBank:MK097140.1)mfa2.1基因的外显子区域,TSP-23插入位点位于两个假定蛋白之间.本研究优化了菰黑粉菌ATMT遗传转化体系,构建了菰黑粉菌T-DNA插入突变体库;筛选到双核菌丝生长缺陷型突变体,并通过基因组重测序的手段明确了相关突变体的T-DNA插入位点,为后续菰黑粉菌二型态转换的调控机理研究奠定了 一定的基础.

The smut fungus Ustilago esculenta obligately parasitizes Zizania latifolia and induces smut galls at the stem tips of host plants. Previous research identified a putative secreted protein, Ue943, which is required for the biotrophic phase of U. esculenta but not for the saprophytic phase. Here, we studied the role of Ue943 during the infection process. Conserved homologs of Ue943 were found in smut fungi. Ue943 can be secreted by U. esculenta and localized to the biotrophic interface between fungi and plants. It is required at the early stage of colonization. The Ue943 deletion mutant caused reactive oxygen species (ROS) production and callose deposition in the host plant at 1 and 5 days post inoculation, which led to failed colonization. The virulence deficiency was restored by overexpressing gene Ue943 or Ue943:GFP. Transcriptome analysis further showed a series of changes in plant hormones following ROS production when the host plant was exposed to ΔUe943. We hypothesize that Ue943 might be responsible for ROS suppression or avoidance of recognition by the plant immune system. The mechanism underlying Ue943 requires further study to provide more insights into the virulence of smut fungi.

为明确双季茭白秋茭的适宜剪叶方式,以双季茭品种浙茭 3 号为试材,于 6 月 15 日定植,以不同离地高度设重剪(离地高 10 cm)、中剪(离地高 25 cm)、轻剪(离地高 40 cm)3 个剪叶处理,以不剪叶为对照,比较分析不同剪叶处理对胡麻斑病、锈病的控制效果及植株分蘖、采茭期、产量等主要性状表现.结果表明,剪叶后植株病情指数显著降低,对胡麻斑病、锈病防治效果分别为 43.95%～49.91%、41.06%～47.69%;重剪、中剪处理提高了植株分蘖数、有效分蘖数及小区产量,净茭率分别显著提高 6.53%、5.58%,一级茭率分别显著提高 5.43%、4.46%.双季茭秋茭以离地高 10～25 cm剪叶为宜.

Indole-3-acetic acid (IAA) belongs to the family of auxin indole derivatives. IAA regulates almost all aspects of plant growth and development, and is one of the most important plant hormones. In microorganisms too, IAA plays an important role in growth, development, and even plant interaction. Therefore, mechanism studies on the biosynthesis and functions of IAA in microorganisms can promote the production and utilization of IAA in agriculture. This mini-review mainly summarizes the biosynthesis pathways that have been reported in microorganisms, including the indole-3-acetamide pathway, indole-3-pyruvate pathway, tryptamine pathway, indole-3-acetonitrile pathway, tryptophan side chain oxidase pathway, and non-tryptophan dependent pathway. Some pathways interact with each other through common key genes to constitute a network of IAA biosynthesis. In addition, functional studies of IAA in microorganisms, divided into three categories, have also been summarized: the effects on microorganisms, the virulence on plants, and the beneficial impacts on plants.

菰黑粉菌(Ustilago esculenta)侵染菰(Zizania latifolia)植株后可诱导植株茎部膨大发育,顶端生长受到显著抑制,这可能与茎部3-吲哚乙酸(3-indoleacetic acid,IAA)极性运输调控相关.本研究克隆获得了6个茭白'浙茭7号'IAA极性运输输出载体相关基因ZlPINs(Z.latifolia PIN formed),这6个基因均具有PIN(PIN formed)蛋白家族的Mem_trans结构域,其中ZlPIN1a、ZlPIN1b、ZlPIN2及ZlPIN3a均与野生稻(Oryza brachyantha)氨基酸的同源性较高,相似度分别高达96.80％、89.27％、90.52％及82.88％.菰植株组织特异性表达分析发现,6个ZlPINs基因均可在根部高表达(P<0.05);在叶片和叶鞘中表达量较低;ZlPIN1a、ZlPIN1b及ZlPIN4可在茎部表达,其中ZlPIN1a表达量显著高于其他ZlPINs基因(P<0.05),推测ZlPIN1a可能是菰植株茎部IAA极性运输的主要调控基因.ZlPIN1a基因(GenBank No.OM782294)克隆序列全长为1770 bp,编码589个氨基酸,有6个外显子和5个内含子;菰黑粉菌体外侵染表明,菰植株茎部ZlPIN1a基因的表达量受菰黑粉菌侵染的抑制调节(P<0.05),正常茭和灰茭植株的膨大发育初期,茎部表达量均显著低于雄茭植株(P<0.05);茭白肉质茎部膨大发育进程中,正常茭茎部ZlPIN1a的表达量先升高后降低,而灰茭茎部的表达量持续升高,ZlPIN1a表达变化可能与菰黑粉菌侵染诱导的2种茎部膨大发育表型相关.本研究筛选获得了菰植株茎部表达的IAA极性运输相关基因ZlPIN1a,明确了菰黑粉菌侵染对其在茎部表达的抑制调节,探讨了其在肉质茎膨大发育中的表达模式,为菰黑粉菌侵染诱导菰植株茎部膨大发育的调节机制研究提供新的思路.

Ustilago esculenta is a smut fungus that obligately infects Zizania latifolia and stimulates tissue swelling to form galls. Unlike T-type, MT-type U. esculenta can only proliferate within plant tissues and infect the offspring of their host. Production of telispores, haploid life, and plant cuticle penetration are not essential for it, which may lead to the degeneration in these processes. Transcriptome changes during the mating of T- and MT-type U. esculenta were studied. The functions of several secreted proteins were further confirmed by knock-out mutants. Our results showed that MT-type U. esculenta can receive environmental signals in mating and circumstance sensing as T-type does. However, MT-type U. esculenta takes a longer time for conjunction tube formation and cytoplasmic fusion. A large number of genes encoding secreted proteins are enriched in the purple co-expression module. They are significantly up-regulated in the late stage of mating in T-type U. esculenta, indicating their relationship with infecting. The knock-out of g6161 (xylanase) resulted in an attenuated symptom. The knock-out of g943 or g4344 (function unidentified) completely blocked the infection at an early stage. This study provides a comprehensive comparison between T- and MT-type during mating and identifies two candidate effectors for further study.

茭白是由菰黑粉菌(Ustilago esculenta)侵染菰(Zizania latifolia)的茎部后形成的可食用肉质茎,在田间生产中常出现灰茭,严重影响茭白的产量和质量.T型菰黑粉菌菌株侵染增殖是引起灰茭的主要因素,正常茭是由MT型菌株侵染形成的,而长期无性繁殖育种使得茭白种苗中T型菌株与MT型菌株共存.因此对茭白种苗中的菌株进行纯化、剔除T型菌株、从而从源头上控制灰茭的产生对茭白产业的发展十分重要.本研究建立了一种T型菌株特异性检测方法,检出限为0.008 8 ng/μL.通过不同育苗方式下种苗中T型菌株的相对含量检测,明确了"带茭"苗的壳里苗为初期纯化的最佳方式,而薹管上段育苗为种苗扩繁的最佳方式.基于T型菌株对逆境的适应力远高于MT型菌株,最终本研究通过对茭白种苗进行5年的逆境处理,结合T型菰黑粉菌检测,获得了一批不产灰茭的纯化茭白种苗.以上结果对茭白种苗的繁育具有理论和实践指导意义.

In this study, a quantum-dot-bead (QB)-based fluorescence-linked immunosorbent assay (FLISA) using nanobodies was established for sensitive determination of the Cry2A toxin in cereal. QBs were used as the fluorescent probe and conjugated with a Cry2A polyclonal antibody. An anti-Cry2A nanobody P2 was expressed and used as the capture antibody. The results revealed that the low detection limit of the developed QB-FLISA was 0.41 ng/mL, which had a 19-times higher sensitivity than the traditional colorimetric ELISA. The proposed assay exhibited a high specificity for the Cry2A toxin, and it had no evident cross-reactions with other Cry toxins. The recoveries of Cry2A from the spiked cereal sample ranged from 86.6–117.3%, with a coefficient of variation lower than 9%. Moreover, sample analysis results of the QB-FLISA and commercial ELISA kit correlated well with each other. These results indicated that the developed QB-FLISA provides a potential approach for the sensitive determination of the Cry2A toxin in cereals.

Cry toxins have been widely used as biopesticides and applied in transgenic crops for the control of pests, raising public concerns for unforeseen environmental and health risks. In this study, five phage-displayed nanobodies against Cry3Bb toxin were selected from a naive nanobody library by screening. Using purified phage-displayed nanobody as capture antibody, a novel double-antibody sandwich fluorescence-linked immunosorbent assay (FLISA) based on quantum dot beads (QBs) was established for the determination of Cry3Bb toxin. The developed method exhibited a linear working range of 31.25-500 ng/mL and a low detection limit of 8.45 ng/mL. The recoveries of Cry3Bb toxin from spiked corn samples were ranging from 93.1% to 116.8% with a coefficient of variation lower than 9.4%. Molecular modeling results showed that key amino acids in the CDR3 region of nanobody P27 combine with Cry3Bb toxin through hydrogen bonds and hydrophobic interactions. These results indicated that the phage-displayed nanobody-based QBs-FLISA may provide a possible tool for detecting Cry3Bb toxin in corn.

为确定高山茭白植株的合理剪叶时期,以单季茭品种美人茭为试材,以15 d为梯度设3个剪叶时期处理,分析不同剪叶时期对茭白锈病、胡麻斑病的防治效果及雄灰茭率、采茭期、产量等主要性状的表现.结果表明,剪叶处理对病害具有显著的控制效果,与不剪叶相比,在药剂防治次数减少2次的情况下,对锈病防治效果为36.20％～48.90％,对胡麻斑病防治效果为22.70％～58.16％,采茭期推迟4～28 d,净茭率提高2.4％～4.7％,有效分蘖数、雄灰茭率及产量无显著差异.综合不同时期剪叶处理对茭白的病害控制效果及主要经济性状分析,高山茭白5月20日剪叶处理效果最佳.

茭白是我国重要水生蔬菜,是菰黑粉菌(Ustilago esculenta)菌丝侵染菰(Zizania latifolia)植株茎部而诱导形成的膨大肉质茎.RPM1(resistance to pseudomonas maculicola 1)作为抗病相关基因,能够识别病原菌的入侵信号并激活植物自身的防卫反应.本研究从茭白提取基因组DNA,采用cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)方法克隆获得2个响应菰黑粉菌侵染的基因ZlRPM1.1(GenBank No.KP729626)和ZlRPM1.2(GenBank No.KP729627),经氨基酸序列比对推测其属于CC-NBS-LRR(coiled coil-nucleotide binding site-leucine rich repeat)类植物抗病相关基因,与栽培水稻(Oryza sativa)的同源基因遗传距离较近,ZlRPM1.1和ZlRPM1.2与栽培水稻的相似性分别为61％和79％.采用菰黑粉菌体外人工侵染菰植株幼苗,对茎部进行qPCR分析的结果显示,外源菰黑粉菌菌丝侵染能够显著诱导菰植株茎部ZlRPM1.1和ZlRPM1.2基因的增强表达.膨大发育初期不同膨大表型茎部的qPCR分析发现,2个ZlRPM1基因在充满菰黑粉菌孢子的灰茭中的表达量均显著高于正常茭白,可能与两种膨大表型茎部中T型与MT型菰黑粉菌菌丝的侵染能力相关.正常茭白茎部膨大发育期间的qPCR分析表明,茎部膨大发育中后期,2个ZlRPM1基因均有显著增强表达,并在茎部膨大发育至茎长15 cm时表达量最高;但ZlRPM1.2基因在茎部8叶期(膨大起始)表达量显著升高,茎部表达量显著高于叶片;而ZlRPM1.1基因在茎部膨大起始时表达量升高不显著,叶片表达量显著高于茎部.上述结果表明,2个ZlRPM1基因均参与菰黑粉菌菌丝侵染诱导的茎部膨大发育调节,其中ZlRPM1.2可能是菰茎部参与响应菰黑粉菌菌丝侵染的重要调节基因.本研究为深入探讨菰植株响应菰黑粉菌菌丝侵染及茎部膨大发育的调节机制提供参考依据.

目的:建立基于特征肽段的新冠病毒刺突蛋白S1亚基定量方法.方法:根据蛋白序列、糖基化修饰位点等筛选特征肽段;利用胰蛋白酶水解蛋白为相对分子质量较小的肽段,并优化反应参数;最后结合同位素稀释质谱法建立刺突蛋白S1亚基定量检测方法,并进行方法学考察.结果:基于特征肽段的同位素稀释质谱法检测样品中S1亚基质量分数为1.007×10-3,扩展不确定度为0.070×10-3,结果准确,与基于氨基酸分析的同位素稀释质谱法检测结果比较无显著差异.特征肽段质量分数在0.47×10-9～1×10-3范围内,回归相关系数R2>0.9999,线性良好.该方法日内与日间精密度均小于5％,检测准确度均在95％ ～105％ 之间,重复性良好.该方法不受基质中其他蛋白的干扰,可溯源至国际单位制,有助于该蛋白绝对定量、标准物质研制等.结论:该方法可行性良好,后期可供参考用于新冠病毒刺突蛋白S1亚基的定量.

茭田水体富营养化和秸秆不合理处置是茭白面源污染形成的主要原因,严重制约茭白产业可持续发展.介绍了茭白水旱间作种植模式的技术操作要点,分析了水旱间作模式在茭田节水、改良土壤、秸秆无害化利用、清洁田园、节本增效等方面的作用,为茭白产业面源污染防治探索新的技术方法.

菰黑粉菌(Ustilago esculenta)是一种二态性真菌,具有侵染菰(Zizania latifolia)植株的能力,并可使植株茎部膨大形成茭白.二型态转换对菰黑粉菌的侵染能力及致病性至关重要.促丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号级联途径和环化腺苷酸介导的蛋白激酶A(cyclic adenosine monophosphate-protein kinase A,cAMP-PKA)信号转导途径是真菌二型态转换的核心.本研究发现,外源添加cAMP后单倍体出现多位点芽殖现象,且融合菌丝生长受到影响.进一步克隆得到了编码菰黑粉菌PKA催化亚基的基因UePkaC(PkaC like protein in Ustilago esculenta)基因(GenBank No.KR870334.1).序列分析结果显示,该基因全长1346 bp,有1个内含子,两个外显子构成大小为1233 bp的开放阅读框,编码410个氨基酸.该氨基酸序列与其他物种中PKA催化亚基的氨基酸序列同源,如大麦坚黑粉菌(U.hordei)、玉米瘤黑粉菌(U.maydis).表达模式分析发现:UePkaC在菌丝融合生长过程中上调表达;对UePkaC缺失突变体表型分析发现其变长且无融合生长能力和致病性.进一步研究发现,UePkaC突变后无法形成接合管,且信息素合成基因表达受到显著抑制,表明UePkaC基因通过调控a基因的表达从而影响接合管的形成与细胞融合.以上结果表明,UePkaC基因在菰黑粉菌单倍体生长及二型态转换中起着关键作用.该研究为探讨菰黑粉菌的致病机理提供了基础资料.

Ustilago esculenta undergoes an endophytic life cycle in Zizania latifolia. It induces the stem of its host to swell, forming the edible galls called jiaobai in China, which are the second most commonly cultivated aquatic vegetable in China. Z. latifolia raised for jiaobai can only reproduce asexually because the U. esculenta infection completely inhibits flowering. The infection and proliferation in the host plants during the formation of edible gall differ from those of conventional pathogens. Previous studies have shown a close relationship between mitogen-activated protein kinase (MAPK) and fungal pathogenesis. In this study, we explored the functional properties of the MAPK UeKpp2. Cross-species complementation assays were carried out, which indicated a functional complementation between the UeKpp2 of U. esculenta and the Kpp2 of Ustilago maydis. Next, UeKpp2 mutants of the UeT14 and the UeT55 sporidia background were generated; these showed an aberrant morphology of budding cells, and attenuated mating and filamentous growth in vitro, in the context of normal pathogenicity. Interestingly, we identified another protein kinase, UeUkc1, which acted downstream of UeKpp2 and may participate in the regulation of cell shape. We also found a defect of filamentous growth in UeKpp2 mutants that was not related to a defect of the induction of mating-type genes but was directly related to a defect in UeRbf1 induction. Overall, our results indicate an important role for UeKpp2 in U. esculenta that is slightly different from those reported for other smut fungi.

菰孕茭是菰黑粉菌(Ustilago esculenta)侵染菰(Zizania latifolia)植株,诱导茎部膨大发育的结果,但其膨大发育的调节机制尚不清楚.本研究克隆获得菰GH3-8(Gretchen Hagen 3-8)基因的全长序列,其基因序列全长为1915 bp,含有2个内含子.cDNA全长为1173 bp,编码390个氨基酸(GenBank No.MH355951),具有1个GH3结构域及1个吲哚乙酸酰胺合成酶结构域,与粳稻(Oryza sativa ssp.japonica)OsGH3-8(GenBank No.XP_015647797.1)相似度较高.孕茭植株中ZlGH3-8表达变化与菰黑粉菌菌丝侵染增殖相关,茎部ZlGH3-8表达量显著高于叶片(P<0.05);茎部膨大初期灰茭ZlGH3-8表达量显著高于正常茭,而雄茭表达量最少(P<0.05).进一步分析发现,正常茭茎部膨大发育期间ZlGH3-8表达量存在显著差异,表明ZlGH3-8可能参与了菰茎部形态发育,可能与孕茭期间植株对菰黑粉菌侵染相关的免疫防御反应下调相关.本研究初步阐明了ZlGH3-8在菰发育过程中的表达响应,为菰茎部膨大发育机制研究提供了基础资料.

Ustilago esculenta is closely related to the smut fungus Ustilago maydis and, in an endophytic-like life in the plant Zizania latifolia, only infects host stems and causes swollen stems to form edible galls called Jiaobai in China. In order to study its different modes of invasion and sites of symptom development from other smut fungi at the molecular level, we first characterized the a and b mating-type loci of U. esculenta. The a loci contained three a mating-type alleles, encoding two pheromones and one pheromone receptor per allele. The pheromone/receptor system controlled the conjugation formation, the initial step of mating, in which each pheromone was specific for recognition by only one mating partner. In addition, there are at least three b alleles identified in U. esculenta, encoding two subunits of heterodimeric homeodomain transcription factors bE and bW, responsible for hyphal growth and invasiveness. Hyphal formation, elongation and invasion after mating of two compatible partners occurred, only when a heterodimer complex was formed by the bE and bW proteins derived from different alleles. We also demonstrated that even with only one paired pheromone-pheromone receptor, the active b locus heterodimer triggered hyphal growth and infection.

茭白是由活体营养型真菌一菰黑粉菌(Ustilago esculenta)侵染菰(Zizania latif olia)后,在菰茎部形成的可食用肉质茎,已成为我国第二大水生蔬菜.传统的茭白种植及育种途径人力、物力投入较大,田间种植的正常茭白常出现品种的退化,还容易产生灰茭和雄茭,严重影响茭白种植的收益.本研究建立并优化了菰黑粉菌人工接种体系,即在25℃光照12h,22℃黑暗12h的温室环境中,以薹管萌发10～20 d的野生菰幼苗茎基部用OD600值为2.0的菰黑粉菌性亲和单倍体菌株UET1和UET2混合菌液注射,孕茭率达到80％以上.进一步通过转录组数据分析筛选出与灰茭中冬孢子发育的干扰因子基因(interfered factors in teliospores development,Itd1) (GenBank No.MK164419),通过同源重组的方法对菰黑粉菌单倍体菌株UET1和UET2进行基因敲除,获得1对冬孢子发育的干扰因子基因缺失菌株UET1△Itd1(CGMCC No.16723)和UET2△Itd1 (CGMCC No.16724),利用优化的人工接种技术侵染野生菰幼苗,获得了可以孕出正常茭白的植株.以上工作可以提供一种新的高效稳定的茭白育种途径,为实现茭白的人工育种提供可靠的理论及技术基础;同时对后续深入研究菰黑粉菌的二型态转换、菌丝生长或冬孢子形成提供理论基础.