The MLA6 coiled-coil, NBS-LRR protein confers AvrMla6-dependent resistance specificity to Blumeria graminis f. sp. hordei in barley and wheat

The barley Mla locus confers multiple resistance specificities to the obligate fungal biotroph, Blumeria (= Erysiphe) graminis f. sp. hordei. Interspersed within the 240 kb Mla complex are three families of resistance gene homologs (RGHs). Probes from the Mla-RGH1 family were used to identify three classes of cDNAs. The first class is predicted to encode a full-length CC-NBS-LRR protein and the other two classes contain alternatively spliced, truncated variants. Utilizing a cosmid that contains a gene corresponding to the full-length candidate cDNA, two single-cell expression assays were used to demonstrate complementation of AvrMla6-dependent, resistance specificity to B. graminis in barley and wheat. The first of these assays was also used to substantiate previous genetic data that the Mla6 allele requires the signaling pathway component, Rar1, for function. Computational analysis of MLA6 and the Rar1-independent, MLA1 protein reveals 91.2% identity and shows that the LRR domain is subject to diversifying selection. Our findings demonstrate that highly related CC-NBS-LRR proteins encoded by alleles of the Mla locus can dictate similar powdery mildew resistance phenotypes yet still require distinct downstream signaling components.     

Disruption of barley immunity to powdery mildew by an in-frame Lys-Leu deletion in the essential protein SGT1

Barley (Hordeum vulgare L.) Mla (Mildew resistance locus a) and its nucleotide-binding, leucine-rich-repeat receptor (NLR) orthologs protect many cereal crops from diseases caused by fungal pathogens. However, large segments of the Mla pathway and its mechanisms remain unknown. To further characterize the molecular interactions required for NLR-based immunity, we used fast-neutron mutagenesis to screen for plants compromised in MLA-mediated response to the powdery mildew fungus, Blumeria graminis f. sp. hordei. One variant, m11526, contained a novel mutation, designated rar3 (required for Mla6 resistance3), that abolishes race-specific resistance conditioned by the Mla6, Mla7, and Mla12 alleles, but does not compromise immunity mediated by Mla1, Mla9, Mla10, and Mla13. This is analogous to, but unique from, the differential requirement of Mla alleles for the co-chaperone Rar1 (required for Mla12 resistance1). We used bulked-segregant-exome capture and fine mapping to delineate the causal mutation to an in-frame Lys-Leu deletion within the SGS domain of SGT1 (Suppressor of G-two allele of Skp1, Sgt1_{ΔKL308–309}), the structural region that interacts with MLA proteins. In nature, mutations to Sgt1 usually cause lethal phenotypes, but here we pinpoint a unique modification that delineates its requirement for some disease resistances, while unaffecting others as well as normal cell processes. Moreover, the data indicate that the requirement of SGT1 for resistance signaling by NLRs can be delimited to single sites on the protein. Further study could distinguish the regions by which pathogen effectors and host proteins interact with SGT1, facilitating precise editing of effector incompatible variants.     

Constitutive disease resistance requires EDS1 in the Arabidopsis mutants cpr1 and cpr6 and is partially EDS1-dependent in cpr5

The systemic acquired resistance (SAR) response in Arabidopsis is characterized by the accumulation of salicylic acid (SA), expression of the pathogenesis-related (PR) genes, and enhanced resistance to virulent bacterial and oomycete pathogens. The cpr (constitutive expressor of PR genes) mutants express all three SAR phenotypes. In addition, cpr5 and cpr6 induce expression of PDF1.2, a defense-related gene associated with activation of the jasmonate/ethylene-mediated resistance pathways. cpr5 also forms spontaneous lesions. In contrast, the eds1 (enhanced disease susceptibility) mutation abolishes race-specific resistance conferred by a major subclass of resistance (R) gene products in response to avirulent pathogens. eds1 plants also exhibit increased susceptibility to virulent pathogens. Epistasis experiments were designed to explore the relationship between the cpr- and EDS1-mediated resistance pathways. We found that a null eds1 mutation suppresses the disease resistance phenotypes of both cpr1 and cpr6. In contrast, eds1 only partially suppresses resistance in cpr5, leading us to conclude that cpr5 expresses both EDS1-dependent and EDS1-independent components of plant disease resistance. Although eds1 does not prevent lesion formation on cpr5 leaves, it alters their appearance and reduces their spread. This phenotypic difference is associated with increased pathogen colonization of cpr5 eds1 plants compared to cpr5. The data allow us to place EDS1 as a necessary downstream component of cpr1- and cpr6-mediated responses, but suggest a more complex relationship between EDS1 and cpr5 in plant defense.     

A barley SKP1‐like protein controls abundance of the susceptibility factor RACB and influences the interaction of barley with the barley powdery mildew fungus

In an increasing number of plant–microbe interactions, it has become evident that the abundance of immunity‐related proteins is controlled by the ubiquitin–26S proteasome system. In the interaction of barley with the biotrophic barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh), the RAC/ROP [RAT SARCOMA‐related C3 botulinum toxin substrate/RAT SARCOMA HOMOLOGUE (RHO) of plants] guanosine triphosphatase (GTPase) HvRACB supports the fungus in a compatible interaction. By contrast, barley HvRBK1, a ROP‐binding receptor‐like cytoplasmic kinase that interacts with and can be activated by constitutively activated HvRACB, limits fungal infection success. We have identified a barley type II S‐phase kinase 1‐associated (SKP1)‐like protein (HvSKP1‐like) as a molecular interactor of HvRBK1. SKP1 proteins are subunits of the SKP1‐cullin 1‐F‐box (SCF)–E3 ubiquitin ligase complex that acts in the specific recognition and ubiquitination of protein substrates for subsequent proteasomal degradation. Transient induced gene silencing of either HvSKP1‐like or HvRBK1 increased protein abundance of constitutively activated HvRACB in barley epidermal cells, whereas abundance of dominant negative RACB only weakly increased. In addition, silencing of HvSKP1‐like enhanced the susceptibility of barley to haustorium establishment by Bgh. In summary, our results suggest that HvSKP1‐like, together with HvRBK1, controls the abundance of HvRACB and, at the same time, modulates the outcome of the barley–Bgh interaction. A possible feedback mechanism from RAC/ROP‐activated HvRBK1 on the susceptibility factor HvRACB is discussed.     

新模式植物短柄二叶草SGT1 和RAR1 基因沉默和蛋白纯化载体构建

短柄二叶草作为一种新型的模式植物,已成为当前研究热点.其具有基因组小、生长周期短、生存环境简单和容易人工转化等重要生理和遗传特性,被认为是一种潜力巨大的人类研究能源和粮食作物的重要模式植物.SGT1和RAR1基因是高度保守的并与植物抗病功能紧密相关的重要基因.本文采用Gateway克隆技术构建了SGT1和RAR1的基因沉默表达载体.阳性克隆载体经PCR、酶切和测序鉴定.为进一步研究SGT1和RAR1互作蛋白及其功能,采用该克隆技术将基因cDNA全长克隆至串联亲和纯化蛋白表达载体pEarleyGate205中,为纯化SGT1和RAR1及其互作蛋白提供了基础.正确的阳性克隆载体经农杆菌介导转化至短柄二叶草Bd21获得转化株,以期进一步研究SGT1和RAR1基因及蛋白互作对于短柄二叶草抗病功能的影响.     

Chemical induction of disease resistance in rice is correlated with the expression of a gene encoding a nucleotide binding site and leucine-rich repeats

Probenazole (3-allyloxy-1,2-benzisothiazole-1,1-dioxide) is an agricultural chemical primarily used to prevent rice blast disease. Probenazole-treated rice acquires resistance to blast fungus irrespective of the rice variety. The chemical is applied prophylactically, and is thought to induce or bolster endogenous plant defenses. However, the mechanisms underlying this effect have not been established. To understand the mode of the chemical's action, we screened for novel probenazole-responsive genes in rice by means of differential display and identified a candidate gene, RPR1. RPR1 contains a nucleotide binding site and leucine-rich repeats, thus sharing structural similarity with known disease resistance genes. The expression of RPR1 in rice can be up-regulated by treatment with chemical inducers of systemic acquired resistance (SAR) and by inoculation with pathogens. RPR1-related sequences in rice varieties seem to be varied in sequence and/or expression, indicating that RPR1 itself is not a crucial factor for induced resistance in rice. However, Southern blot analysis revealed the existence of homologous sequences in all varieties examined. While the role of RPR1 has yet to be clarified, this is the first report of the identification of a member of this gene class and its induction during the systemic expression of induced disease resistance.     

A phospho-proteomic screen identifies novel S6K1 and mTORC1 substrates revealing additional complexity in the signaling network regulating cell growth

S6K1, a critical downstream substrate of mTORC1, has been implicated in regulating protein synthesis and a variety of processes that impinge upon cell growth and proliferation. While the role of the cytoplasmic p70^S6K1 isoform in the regulation of translation has been intensively studied, the targets and function of the nuclear p85^S6K1 isoform remain unclear. Therefore, we carried out a phospho-proteomic screen to identify novel p85^S6K1 substrates. Four novel putative p85^S6K1 substrates, GRP75, CCTβ, PGK1 and RACK1, and two mTORC1 substrates, ANXA4 and PSMA6 were identified, with diverse roles in chaperone function, ribosome maturation, metabolism, vesicle trafficking and the proteasome, respectively. The chaperonin subunit CCTβ was further investigated and the site of phosphorylation mapped to serine 260, a site located in the chaperonin apical domain. Consistent with this domain being involved in folding substrate interactions, we found that phosphorylation of serine 260 modulates chaperonin folding activity.     

mTOR/S6K1信号通路与胰岛素抵抗的关系及有氧运动对其影响的研究

目的:通过研究高脂饮食和有氧运动对胰岛素抵抗(IR)小鼠骨骼肌雷帕霉素靶蛋白/核糖体S6激酶1(mTOR/S6K1)通路的影响,试图为运动防治IR提供理论依据。方法:8周C57BL/6小鼠随机分为正常饮食组和高脂饮食组,每组各20只,高脂饮食组喂养8周后建立IR模型。随后将正常饮食组再次随机分为正常饮食安静组(NC)和正常饮食运动组(NE);高脂饮食组也随机分为高脂饮食安静组(HC)和高脂饮食运动组(HE)。各运动组进行为期6周、75%VO2max强度跑台训练,每天1次,每次60min,每周5次。实验结束后采用OGTT检测葡萄糖耐量,组织学检测胰岛形态变化,ELISA法检测血清空腹胰岛素水平,Northern blot、Western blot检测骨骼肌中mTOR和S6K1 mRNA和蛋白及其磷酸化蛋白pS6K1-Thr389的表达。结果:与NC组相比,HC组小鼠体重、空腹血清胰岛素值和胰岛β细胞团面积百分比均呈显著增加,且OGTT曲线显示糖耐量明显受损,然而6周有氧运动后以上各指标呈显著性降低,葡萄糖耐量也得到明显改善;且骨骼肌中mTOR、S6K1、pS6K1-Thr389 mRNA和蛋白表达均明显降低。结论:mTOR/S6K1信号通路与高脂饮食诱导IR的发生密切相关,有氧运动明显增加了机体组织对胰岛素的敏感性,推测有氧运动可能通过抑制mTOR/S6K1信号通路,增加IR小鼠骨骼肌的能量代谢从而改善IR。     

The broad-spectrum potato cyst nematode resistance gene (Hero) from tomato is the only member of a large gene family of NBS-LRR genes with an unusual amino acid repeat in the LRR region

The Hero gene of tomato is a broad spectrum resistance gene that confers a high level of resistance to all pathotypes of the potato cyst nematodes Globodera rostochiensis and partial resistance to G. pallida. The gene was identified by map-based cloning, sequencing and complementation analysis of two susceptible tomato lines with an array of 13 overlapping cosmids spanning a total distance of 135 kb. Hero encodes a protein with a nucleotide-binding site (NBS) and a leucine-rich-repeat (LRR) domain and is a member of a gene family of 14 highly homologous genes, which are clustered within a continuous 118-kb region. The isolated Hero gene displayed resistance to various G. rostochiensis pathotypes and partial resistance to G. pallida pathotype Pa2/3 in transgenic tomato lines. None of the Hero homologues conferred resistance to G. rostochiensis pathotypes. Hero can be distinguished from its homologues by the length of a compound hexanucleotide microsatellite, which codes for a charged and repetitive amino acid domain within the LRR. We propose that the expansion of this microsatellite may be involved in the evolution of the Hero resistance gene.     

CRISPR/SpCas9-mediated double knockout of barley Microrchidia MORC1 and MORC6a reveals their strong involvement in plant immunity,transcriptional gene silencing and plant growth

The Microrchidia (MORC) family proteins are important nuclear regulators in both animals and plants with critical roles in epigenetic gene silencing and genome stabilization. In the crop plant barley (Hordeum vulgare), seven MORC gene family members have been described. While barley HvMORC1 has been functionally characterized, very little information is available about other HvMORC paralogs. In this study, we elucidate the role of HvMORC6a and its potential interactors in regulating plant immunity via analysis of CRISPR/SpCas9-mediated single and double knockout (dKO) mutants, hvmorc1 (previously generated and characterized by our group), hvmorc6a, and hvmorc1/6a. For generation of hvmorc1/6a, we utilized two different strategies: (i) successive Agrobacterium-mediated transformation of homozygous single mutants, hvmorc1 and hvmorc6a, with the respective second construct, and (ii) simultaneous transformation with both hvmorc1 and hvmorc6a CRISPR/SpCas9 constructs. Total mutation efficiency in transformed homozygous single mutants ranged from 80 to 90%, while upon simultaneous transformation, SpCas9-induced mutation in both HvMORC1 and HvMORC6a genes was observed in 58% of T0 plants. Subsequent infection assays showed that HvMORC6a covers a key role in resistance to biotrophic (Blumeria graminis) and necrotrophic (Fusarium graminearum) plant pathogenic fungi, where the dKO hvmorc1/6a showed the strongest resistant phenotype. Consistent with this, the dKO showed highest levels of basal PR gene expression and derepression of TEs. Finally, we demonstrate that HvMORC1 and HvMORC6a form distinct nucleocytoplasmic homo-/heteromers with other HvMORCs and interact with components of the RNA-directed DNA methylation (RdDM) pathway, further substantiating that MORC proteins are involved in the regulation of TEs in barley.     

The disease resistance gene Dm3 is infrequent in natural populations of Lactuca serriola due to deletions and frequent gene conversions at the RGC2 locus

Resistance genes can exhibit heterogeneous patterns of variation. However, there are few data on their frequency and variation in natural populations. We analysed the frequency and variation of the resistance gene Dm3, which confers resistance to Bremia lactucae (downy mildew) in 1033 accessions of Lactuca serriola (prickly lettuce) from 49 natural populations. Inoculations with an isolate of Bremia lactucae carrying avirulence gene Avr3 indicated that the frequency of Dm3 in natural populations of L. serriola was very low. Molecular analysis demonstrated that Dm3 was present in only one of the 1033 wild accessions analysed. The sequence of the 5' region of Dm3 was either highly conserved among accessions, or absent. In contrast, frequent chimeras were detected in the 3' leucine-rich repeat-encoding region. Therefore low frequency of the Dm3 specificity in natural populations was due to either the recent evolution of Dm3 specificity, or deletions of the whole gene as well as variation in 3' region caused by frequent gene conversions. This is the most extensive analysis of the prevalence of a known disease resistance gene to date, and indicates that the total number of resistance genes in a species may be very high. This has implications for the scales of germplasm conservation and exploitation of sources of resistance.     

转小麦冷胁迫蛋白截短基因烟草及其抗病性分析

TA3-13是克隆于小麦冷胁迫蛋白基因的截短片段。原核表达的TA3-13蛋白能够诱导烟草产生显著的抗烟草花叶病毒(TMV)的作用。文章将TA3-13基因片段克隆到植物表达载体pBI121上,构建成转基因重组体pB-3-13,通过冻融法转化农杆菌EHA105,构建成转基因侵染菌株。采用叶盘法将pB-3-13转化三生烟草,经卡那霉素抗性筛选,获得48株T0代再生植株。通过PCR检测,鉴定出33株转基因单株,收获了20株种子作为T1代株系。PCR-Southern杂交结果显示,PCR阳性条带与TA3-13探针有特异性杂交,说明外源基因被转化到烟草的基因组中。选取两个T1代株系的烟草植株用于各项测定。GUS组织化学活性鉴定和RT-PCR检测结果显示,外源基因可以成功地表达。接种TMV病毒后,转基因烟草抗TMV的能力较转空载体烟草提高3～5倍。转基因烟草具有抗TMV侵入和抗病毒病害发展的作用,同时转基因烟草可以抗细菌软腐病菌的扩展。     

Capillary electrophoresis-based profiling and quantitation of total salicylic acid and related phenolics for analysis of early signaling in Arabidopsis disease resistance

A capillary electrophoresis-based method for quantitation of total salicylic acid levels in Arabidopsis leaves was developed. Direct comparison to previous high-performance liquid chromatography (HPLC)-based measurements showed similar levels of salicylic acid. Simultaneous quantitation of trans-cinnamic acid, benzoic acid, sinapic acid, and an internal recovery standard was achieved. A rapid, streamlined protocol with requirements for plant tissue reduced relative to those of HPLC-based protocols is presented. Complicated, multiparameter experiments were thus possible despite the labor-intensive nature of inoculating plants with bacterial pathogens. As an example of this sort of experiment, detailed time course studies of total salicylic acid accumulation by wild-type Arabidopsis and two lines with mutations affecting salicylic acid accumulation in response to either of two avirulent bacterial strains were performed. Accumulation in the first 12h was biphasic. The first phase was partially SID2 and NDR1 dependent with both bacterial strains. The second phase was largely independent of both genes with bacteria carrying avrB, but dependent upon both genes with bacteria carrying avrRpt2. Virulent bacteria did not elicit salicylic acid accumulation at these time points. Application of this method to various Arabidopsis pathosystems and the wealth of available disease resistance signaling mutants will refine knowledge of disease resistance and associated signal transduction.     

澳门地区褐家鼠对溴鼠灵的抗性检测及其Vkorc1基因多态性分析

第二代抗凝血灭鼠剂因比第一代抗凝血灭鼠剂有更好的灭杀效果和安全性而被广泛使用,但长期使用同样也存在引发鼠类抗性的隐患。近年,学者已发现了第二代抗凝血灭鼠剂溴敌隆 (bromadiolone) 和鼠得克 (difenacoum) 的抗性鼠。溴鼠灵 (brodifacoum) 是一种目前在国内广泛使用且毒性极强的第二代抗凝血灭鼠剂,能引发鼠类凝血功能障碍和细胞毒性,但对于溴鼠灵是否已驱动鼠类发生抗性进化尚不清楚。澳门地区从1995年开始持续使用溴鼠灵,为研究鼠类对溴鼠灵的抗药性进化提供了良好模型。本研究于2019年10—12月在澳门地区共捕获61只褐家鼠,对其中44只开展了LFP实验 (0.005%溴鼠灵),实验鼠的平均摄毒量为 (15.28 ± 1.40) mg/kg,在7天内全部死亡。此外,对全部61只褐家鼠的维生素K环氧化物还原酶复合体亚单位1 (vitamin K-epoxide reductas complex 1, Vkorc1) 的基因序列测定表明,在Vkorc1基因外显子的已知抗药关联位点处未检测到非同义核苷酸突变,但检测到2个同义核苷酸突变 [第68位氨基酸：H (CAC)-H (CAT),突变发生率为100%;第82位氨基酸：I (ATA)-I (ATT),突变发生率为32.72%]。该研究表明,使用溴鼠灵25年后,澳门地区褐家鼠虽没有产生群体抗药性,但仍需持续监测抗药性的发生和发展。     

Single residues in the LRR domain of the wheat PM3A immune receptor can control the strength and the spectrum of the immune response

The development of improved plant nucleotide‐binding, leucine‐rich repeat (LRR) immune receptors (NLRs) has mostly been based on random mutagenesis or on structural information available for specific receptors complexed with the recognized pathogen effector. Here, we use a targeted mutagenesis approach based on the natural diversity of the Pm3 powdery mildew resistance alleles present in different wheat (Triticum aestivum) genotypes. In order to understand the functional importance of the amino acid polymorphisms between the active immune receptor PM3A and the inactive ancestral variant PM3CS, we exchanged polymorphic regions and residues in the LRR domain of PM3A with the corresponding segments of PM3CS. These novel variants were functionally tested for recognition of the corresponding AVRPM3^A2/F2 avirulence protein in Nicotiana benthamiana. We identified polymorphic residues in four regions of PM3A that enhance the immune response, but also residues that reduce it or result in complete loss of function. We found that the identified critical residues in PM3A modify its activation threshold towards different protein variants of AVRPM3^A2/F2. PM3A variants with a lowered threshold gave a stronger overall response and gained an extended recognition spectrum. One of these variant proteins with a single amino acid change was stably transformed into wheat, where it conferred race‐specific resistance to mildew. This is a proof of concept that improved PM3A variants with an enlarged recognition spectrum can be engineered based on natural diversity by exchanging single or multiple residues that modulate resistance function.