A G-type lectin receptor-like kinase regulates the perception of oomycete apoplastic expansin-like proteins

Phytophthora capsici is one of the most harmful pathogens in agriculture, which threatens the safe production of multiple crops and causes serious economic losses worldwide. Here, we identified a P. capsici expansin-like protein, PcEXLX1, by liquid chromatography-tandem mass spectrometry from Nicotiana benthamiana apoplastic fluid infected with P. capsici. Clustered regularly interspaced short palindromic repeats/crispr associated protein 9 (CRISPR/Cas9)-mediated PcEXLX1 knockout mutants exhibited significantly enhanced virulence, while the overexpression of PcEXLX1 impaired the virulence. Prokaryotically expressed PcEXLX1 activated multiple plant immune responses, which were BRI1-associated kinase 1 (BAK1)- and suppressor of BIR1-1 (SOBIR1)-dependent. Furthermore, overexpression of PcEXLX1 homologs in N. benthamiana could also increase plant resistance to P. capsici. A G-type lectin receptor-like kinase from N. benthamiana, expansin-regulating kinase 1 (ERK1), was shown to regulate the perception of PcEXLX1 and positively mediate the plant resistance to P. capsici. These results reveal that the expansin-like protein, PcEXLX1, is a novel apoplastic effector with plant immunity-inducing activity of oomycetes, perception of which is regulated by the receptor-like kinase, ERK1.     

Nicotiana benthamiana LRR‐RLP NbEIX2 mediates the perception of an EIX‐like protein from Verticillium dahliae

Verticillium wilt diseases caused by the soil‐borne fungus Verticillium dahliae result in devastating yield losses in many economically important crops annually. Here, we identified a novel ethylene‐inducing xylanase (EIX)‐like protein, VdEIX3, from V. dahliae, which exhibits immunity‐inducing activity in Nicotiana benthamiana. In vitro‐purified VdEIX3 can induce strong oxidative burst, activate the expression of defense‐related genes, and increase resistance against oomycete and fungal pathogens in N. benthamiana. VdEIX3 orthologs of other Verticillium pathogens also induce cell death in N. benthamiana, which form a new type of EIX protein family that is distinct from the known EIX proteins. A leucine‐rich repeat receptor‐like protein, NbEIX2, regulates the perception of VdEIX3 in N. benthamiana. Our results demonstrate that VdEIX3 is a novel EIX‐like protein that can be recognized by N. benthamiana NbEIX2, and also suggest that NbEIX2 is a promising receptor‐like protein that is potentially applicable to transgenic breeding for improving resistance to Verticillium wilt diseases.     

Transformation of callus cultures of nine plant species mediated by agrobacterium

A simple method for the Agrobacterium-mediated transformation of callus cultures of nine plant species, Lycopersicum esculentum Mill, Petunia hybrida Vilm, Pimpinella anisum L., Solanum melongena L., S. tuberosum L., Nicotiana glauca Graham, N. glutinosa L., N. plumbaginifolia Viviani and N. tabacum L., is described. Plant calli were resuspended in liquid media, co-cultivated with A. tumefaciens, and plated on restrictive media. The combination of a gene for kanamycin resistance and a gene for firefly luciferase was convenient in the selection and confirmation of hundreds of transformants. Four strains of A. tumefaciens, A208, A348, A281, and PC2760, were employed. All of the callus cultures were successfully transformed with at least one strain of A. tumefaciens, and A281 was the most effective of the four strains. N. glutinosa, N. plumbaginifolia, N. tabacum, P. hybrida and L. esculentum were transformed more efficiently than the other species tested.     

Revision of the genus Noculacia Mayer, 1903 (Crustacea: Amphipoda: Caprellidea) with the description of two new species

The genus Noculacia Mayer, 1903 is reviewed. Two new species, N. africana n. sp. and N. australiensis n. sp., are described based on material collected from southeast Africa and western-southern Australia, respectively. Noculacia bullata Mayer, 1903, the type species of the genus, is redescribed. Noculacia bogisa Mayer, 1903 is transferred to the genus Pseudoprotella Mayer, 1890 mainly on the basis of the presence of a well developed molar, the structure of pereopods 3 and 4, and the setal formula of the mandibular palp being 2-x-1. The genus Noculacia is presently composed of three species: N. africana n. sp., N. australiensis n. sp, and N. bullata Mayer, 1903. The genus Pseudoprotella is composed of P. bogisa (Mayer, 1903), P. inermis Chevreux, 1927, and P. phasma (Montagu, 1804).     

The Phytophthora effector Avh94 manipulates host jasmonic acid signaling to promote infection

The oomycete pathogen Phytophthora sojae is a causal agent of soybean root rot. Upon colonization of soybeans, P. sojae secretes various RXLR effectors to suppress host immune responses, supporting successful infection. Previous research has demonstrated that the RXLR effector Avh94 functions as a virulence effector, but the molecular mechanism underlying its role in virulence remains unknown. Here, we demonstrate that Avh94 overexpression in plants and pathogens promotes Phytophthora infection. Avh94 interacts with soybean JAZ1/2, which is a repressor of jasmonic acid (JA) signaling. Avh94 stabilizes JAZ1/2 to inhibit JA signaling and silencing of JAZ1/2 enhances soybean resistance against P. sojae. Moreover, P. sojae lines overexpressing Avh94 inhibit JA signaling. Furthermore, exogenous application of methyl jasmonate improves plant resistance to Phytophthora. Taken together, these findings suggest that P. sojae employs an RXLR effector to hijack JA signaling and thereby promote infection.     

The calcium‐dependent protein kinase ZmCDPK7 functions in heat‐stress tolerance in maize

Global warming poses a serious threat to crops. Calcium‐dependent protein kinases (CDPKs)/CPKs play vital roles in plant stress responses, but their exact roles in plant thermotolerance remains elusive. Here, we explored the roles of heat‐induced ZmCDPK7 in thermotolerance in maize. ZmCDPK7‐overexpressing maize plants displayed higher thermotolerance, photosynthetic rates, and antioxidant enzyme activity but lower H₂O₂ and malondialdehyde (MDA) contents than wild‐type plants under heat stress. ZmCDPK7‐knockdown plants displayed the opposite patterns. ZmCDPK7 is attached to the plasma membrane but can translocate to the cytosol under heat stress. ZmCDPK7 interacts with the small heat shock protein sHSP17.4, phosphorylates sHSP17.4 at Ser‐44 and the respiratory burst oxidase homolog RBOHB at Ser‐99, and upregulates their expression. Site‐directed mutagenesis of sHSP17.4 to generate a Ser‐44‐Ala substitution reduced ZmCDPK7's enhancement of catalase activity but enhanced ZmCDPK7's suppression of MDA accumulation in heat‐stressed maize protoplasts. sHSP17.4, ZmCDPK7, and RBOHB were less strongly upregulated in response to heat stress in the abscisic acid‐deficient mutant vp5 versus the wild type. Pretreatment with an RBOH inhibitor suppressed sHSP17.4 and ZmCDPK7 expression. Therefore, abscisic acid‐induced ZmCDPK7 functions both upstream and downstream of RBOH and participates in thermotolerance in maize by mediating the phosphorylation of sHSP17.4, which might be essential for its chaperone function.     

Fg12 ribonuclease secretion contributes to Fusarium graminearum virulence and induces plant cell death

Filamentous fungal pathogens secrete effectors that modulate host immunity and facilitate infection. Fusarium graminearum is an important plant pathogen responsible for various devastating diseases. However, little is known about the function of effector proteins secreted by F. graminearum. Herein, we identified several effector candidates in the F. graminearum secretome. Among them, the secreted ribonuclease Fg12 was highly upregulated during the early stages of F. graminearum infection in soybean; its deletion compromised the virulence of F. graminearum. Transient expression of Fg12 in Nicotiana benthamiana induced cell death in a light‐dependent manner. Fg12 possessed ribonuclease (RNase) activity, degrading total RNA. The enzymatic activity of Fg12 was required for its cell death‐promoting effects. Importantly, the ability of Fg12 to induce cell death was independent of BAK1/SOBIR1, and treatment of soybean with recombinant Fg12 protein induced resistance to various pathogens, including F. graminearum and Phytophthora sojae. Overall, our results provide evidence that RNase effectors not only contribute to pathogen virulence but also induce plant cell death.     

烟草乙烯转录因子ERF基因NtTOE3的克隆、载体构建及表达分析

ERF（Ethylene-responsive factor）是一类具有AP2特征结构域的乙烯应答转录因子,在响应植物的逆境胁迫应答中起关键作用。为明确ERF家族成员TOE3在烟草中的生物学功能和调控机制,同源克隆普通烟草K326中NtTOE3基因cDNA全长,利用实时荧光定量PCR（qRT-PCR）表征基因表达模式,结合生物信息学分析,对基因功能和调控机制进行初步预测。结果表明,该基因全长1 356 bp,编码451个氨基酸残基,预测分子量为49.84 ku。生物信息学分析表明,该蛋白是一个亲水蛋白,可能定位在细胞核,且与美花烟草（Nicotiana sylvestris）NsTOE3有96%的同源性,故命名为NtTOE3。组织表达分析发现,该基因在烟草根、茎、叶、花中均有表达,其中茎中表达量最高。逆境胁迫试验表明,该基因能快速响应低钾、高盐、干旱、H₂O₂、ABA和4℃等逆境处理。表明NtTOE3转录因子在烟草非生物胁迫中起着重要调节作用。     

生长阶段及环境因子对东海原甲藻氨基酸氧化酶活性的影响

以中国沿海典型赤潮藻东海原甲藻为实验材料, 研究了不同生长阶段以及温度、光照和氮源对其氨基酸氧化酶活性的影响。结果表明, 在缺氮条件下东海原甲藻显示较高的氨基酸氧化酶活性。在15-30℃温度范围均检测出氨基酸氧化酶活性, 较低温度下(15-20℃)的氨基酸氧化酶活性显著高于高温处理组(25-30℃)(p<0.05), 其中20℃时的酶活最高。在50-100 μmol/(m²·s)的光强下, 氨基酸氧化酶活性较高(0.38-0.47 fmol/(h·cell)), 而在2 μmol/(m²·s)的低光强下, 虽然酶活受到显著抑制, 但仍达到0.28 fmol/(h·cell)。氮源组成对氨基酸氧化酶活性具有显著影响, 以丙氨酸为唯一氮源时的酶活最高(0.44 fmol/(h·cell)), NH₄⁺+丙氨酸为氮源时的酶活最低(0.22 fmol/(h·cell))。研究显示, 光照、温度和氮源是东海原甲藻氨基酸氧化酶活性的关键调控因子。东海原甲藻不仅能够有效利用游离氨基酸, 而且适应较广的温度和较低的光照条件, 这可能是东海原甲藻赤潮形成和持续的重要原因。     

Ethylene production in Botrytis cinerea‐ and oligogalacturonide‐induced immunity requires calcium‐dependent protein kinases

Plant immunity against pathogens is achieved through rapid activation of defense responses that occur upon sensing of microbe‐ or damage‐associated molecular patterns, respectively referred to as MAMPs and DAMPs. Oligogalacturonides (OGs), linear fragments derived from homogalacturonan hydrolysis by pathogen‐secreted cell wall‐degrading enzymes, and flg22, a 22‐amino acid peptide derived from the bacterial flagellin, represent prototypical DAMPs and MAMPs, respectively. Both types of molecules induce protection against infections. In plants, like in animals, calcium is a second messenger that mediates responses to biotic stresses by activating calcium‐binding proteins. Here we show that simultaneous loss of calcium‐dependent protein kinases CPK5, CPK6 and CPK11 affects Arabidopsis thaliana basal as well as elicitor‐ induced resistance to the necrotroph Botrytis cinerea, by affecting pathogen‐induced ethylene production and accumulation of the ethylene biosynthetic enzymes 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthase 2 (ACS2) and 6 (ACS6). Moreover, ethylene signaling contributes to OG‐triggered immunity activation, and lack of CPK5, CPK6 and CPK11 affects the duration of OG‐ and flg22‐induced gene expression, indicating that these kinases are shared elements of both DAMP and MAMP signaling pathways.     

毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸*

目的:改造毕赤酵母使其异源合成类黄酮生物合成途径的重要中间体肉桂酸、对香豆酸,并优化前体芳香族氨基酸生物合成途径以提高毕赤酵母的生产能力。方法:在毕赤酵母GS115中利用乙醇诱导型人工转录系统表达Rhodotorula glutinis来源的苯丙氨酸解氨酶,并在该重组菌株中分别过表达胞内芳香族氨基酸生物合成途径中的关键酶或其突变体以进行优化。结果:异源表达苯丙氨酸解氨酶可使毕赤酵母将自身产生的L-苯丙氨酸、L-酪氨酸转化为肉桂酸(38.8 mg/L)、对香豆酸(34.2 mg/L),而通过过表达相关酶进行优化,最终肉桂酸和对香豆酸的产量分别达到124.1 mg/L和302.0 mg/L。结论:利用新的异源宿主毕赤酵母成功合成了肉桂酸、对香豆酸,并对胞内的芳香族氨基酸生物合成途径进行了优化,表明毕赤酵母具有生产黄酮类化合物的应用潜力,也为其他芳香族氨基酸衍生物或植物化合物在毕赤酵母中的异源合成奠定了基础。     

Trehalose phosphate synthase 5‐dependent trehalose metabolism modulates basal defense responses in Arabidopsis thaliana

Despite the recent discovery that trehalose synthesis is important for plant development and abiotic stress tolerance, the effects of trehalose on biotic stress responses remain relatively unknown. In this study, we demonstrate that TREHALOSE PHOSPHATE SYNTHASE 5 (TPS5)-dependent trehalose metabolism regulates Arabidopsis thaliana defenses against pathogens (necrotrophic Botrytis cinerea and biotrophic Pseudomonas syringae). Pathogen infection increased trehalose levels and upregulated TPS5 expression. Application of exogenous trehalose significantly improved plant defenses against B. cinerea, but increased the susceptibility of plants to P. syringae. We demonstrate that elevated trehalose biosynthesis, in transgenic plants over-expressing TPS5, also increased the susceptibility to P. syringae, but decreased the disease symptoms caused by B. cinerea. The knockout of TPS5 prevented the accumulation of trehalose and enhanced defense responses against P. syringae. Additionally, we observed that a TPS5-interacting protein (multiprotein bridging factor 1c) was required for induced expression of TPS5 during pathogen infections. Furthermore, we show that trehalose promotes P. syringae growth and disease development, via a mechanism involving suppression of the plant defense gene, Pathogenesis-Related Protein 1. These findings provide insight into the function of TPS5-dependent trehalose metabolism in plant basal defense responses.     

苹果黑腐皮壳菌CAP超家族蛋白VmPR1c的降解功能域和降解途径

真菌与寄主互作过程中常分泌多种效应蛋白,但植物病原真菌CAP超家族蛋白是否参与致病过程尚不明确。基于苹果黑腐皮壳菌Valsa mali基因组CAP同源基因研究发现VmPR1c敲除后突变体致病性明显下降,但VmPR1c蛋白在表达过程中被降解。本研究借助BLAST、NCBI CDD web server、SignalP 4.1、TMHMM 2.0等进行序列分析,利用缺失突变法及在蛋白表达过程中加蛋白酶抑制剂分别对该蛋白的降解功能域和降解途径进行了探索。C端区段缺失突变结果显示,突变涉及CAP结构域中的α-helix结构和CBM区域时,Western blot条带呈现明显加深;涉及CTE区域及CAP结构域中的半胱氨酸时,Western blot条带则呈现不同程度地减弱。替换VmPR1c的信号肽或突变其信号肽切割位点序列,Western blot条带均有不同程度地加深。分别对CTE和NTE中的脯氨酸进行点突变后,蛋白降解更加严重。在蛋白表达过程中加入蛋白酶体抑制剂MG132和溶酶体抑制剂chloroquine,与对照VmPR1c ^△SP-GFP相比,Western blot结果无明显变化,表明VmPR1c蛋白序列C端区域中的CTE、信号肽及其切割位点序列以及CAP结构域中的α-helix结构介导其降解,其中CTE和NTE中的脯氨酸对该蛋白具保护作用。此外,VmPR1c的降解不通过蛋白酶体和溶酶体途径。     

Pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1R) co-localizes with activity-dependent neuroprotective protein (ADNP) in the mouse brains

The crustacean hyperglycemic hormone is the most abundant neuropeptide present in the eyestalk of Crustacea and its main role is to control the glucose level in the hemolymph. Our study was aimed at assessing the importance of C-terminal amidation for its biological activity. Two recombinant peptides were produced, Asl-rcHH-Gly with a free carboxyl terminus and Asl-rcHH-amide with an amidated C-terminus. Homologous bioassays performed on the astacid crayfish Astacus leptodactylus showed that the amidated peptide had a stronger hyperglycemic effect compared to the non-amidated peptide. To assess the relevance of amidation also in other decapods and how much the differences in the cHH amino acid sequence can affect the functionality of the peptides, we carried out heterologous bioassays on the cambarid Procambarus clarkii and palaemonid Palaemon elegans. The Asl-rcHH-amide elicited a good response in P. clarkii and in P. elegans. The injection of Asl-rcHH-Gly evoked a weak response in both species. These results prove the importance of C-terminal amidation for the biological activity of cHH in crayfish as well as the role of the peptide primary sequence for the species-specificity hormone-receptor recognition.