A Volatile Substance, β-Caryophyllene, from Talaromyces wortmannii Promotes Growth and Tolerance to Diseases on Several Plants

A plant growth-promoting fungus, Talaromyces wortmannii strain FS2 was isolated from an agricultural field at Okayama Pref. FS2 enhanced seed germination, root elongation and leaf growth of Brassica rapa var perviridis (Komatsuna). Such plant growth-promoting effect was observed in the same sealed chamber where FS2 was cultured on PDA medium separated from seedlings, suggesting effective volatile compound(s). GC‒MS analysis showed that FS2 emitted at least seven terpenoids, of which a volatile was identified as β‒caryophyllene. β‒caryophyllene alone promoted the growth of cucumber, Nicotiana benthamiana and barley. Furthermore β‒caryophyllene increased the yield of cucumber fruits. Interestingly, we found that β‒caryophyllene conditioned these plants to be resistant to respective diseases caused by Colletotrichum orbiculare, Botrytis cinerea or Blumeria graminis f. sp hordei. The findings indicate that β‒caryophyllene has desirable dual features and therefore, it is available to cultivation of many crops.岡山県総社市の圃場から分離した植物生育促進菌Talaromyces wortmannii FS2が生産するβ-caryophylleneは，コマツナ（アブラナ科）のみならず，キュウリ（ウリ科），タバコ（ナス科）およびオオムギ（イネ科）など広汎な植物に対して，生育促進作用および耐病性増進作用を示したことから，有用な農業資材として利用可能であるものと考察した

Isolation and Identification of a Plant Growth-Promoting Fungus from an Agricultural Field in Okayama Prefecture

A plant growth-promoting fungus was isolated from an agricultural field in Okayama Prefecture, Japan. The strain FS2, which enhanced seed germination, root elongation and leaf growth of Brassica rapa var. perviridis, was identified as Talaromyces wortmannii based on ITS1 sequence and its morphology.本研究では，実際の生産圃場から植物生育促進菌（PGPF）の探索を試み，コマツナの生育を促進するFS2株を分離した．FS2株の形態観察並びにのITS1領域の系統樹解析から本菌をTalaromyces wortmanniiと同定した

Efficacy and safety of luseogliflozin added to various oral antidiabetic drugs in Japanese patients with type 2 diabetes mellitus

Introduction: Two studies were carried out to investigate the efficacy and safety of luseogliflozin added to existing oral antidiabetic drugs (OADs) in Japanese type 2 diabetic patients inadequately controlled with OAD monotherapy. Materials and Methods: In the trial involving add‐on to sulfonylureas (study 03‐1), patients were randomly assigned to receive luseogliflozin 2.5 mg or a placebo for a 24‐week double‐blind period, followed by a 28‐week open‐label period. In the open‐label trial involving add‐on to other OADs; that is, biguanides, dipeptidyl peptidase‐4 inhibitors, thiazolidinediones, glinides and α‐glucosidase inhibitors (study 03‐2), patients received luseogliflozin for 52 weeks. Results: In study 03‐1, luseogliflozin significantly decreased glycated hemoglobin at the end of the 24‐week double‐blind period compared with the placebo (–0.88%, P < 0.001), and glycated hemoglobin reduction from baseline at week 52 was –0.63%. In study 03‐2, luseogliflozin added to other OADs significantly decreased glycated hemoglobin from baseline at week 52 (–0.52 to –0.68%, P < 0.001 for all OADs). Bodyweight reduction was observed in all add‐on therapies, even with agents associated with weight gain, such as sulfonylureas and thiazolidinediones. Most adverse events were mild in severity. When added to a sulfonylurea, incidences of hypoglycemia during the double‐blind period were 8.7% and 4.2% for luseogliflozin and placebo, respectively, but no major hypoglycemic episodes occurred. The frequency and incidences of adverse events of special interest for sodium glucose cotransporter 2 inhibitors and adverse events associated with combined OADs were acceptable. Conclusions: Add‐on therapies of luseogliflozin to existing OADs improved glycemic control, reduced bodyweight and were well tolerated in Japanese type 2 diabetic patients. These trials were registered with the Japan Pharmaceutical Information Center (add on to sulfonylurea: JapicCTI‐111507; add on to other OADs: JapicCTI‐111508)

エンドウのエリシター誘導性遺伝子発現におけるAAAGモチーフとPsDof1タンパク質の関与

Recently, we, isolated cDNA clone, PsDof1, from clicitor-treated pea cDNA library. The putative gene product, a PsDof1, encodes DNA binding protein that specifically binds the DNA fragment containing AAAG core sequence. In this paper we report that GST-PsDof1 fusion protein specifically binds to the promoter region containing AAAG core sequence(s) of PsCHS1, one of the elicitor-inducible genes encoding chalcone synthase (CHS). Furthermore the addition of DNA fragment containing AAAG motif to the 35S minimal promoter provided the elicitor-responsibility in transient transfection assay using pea protoplasts. These results suggest that PsDof1 might be involved in defense responses by acitivating the transcription by a binding to AAAG core sequence in the promoter of the defense-related genes in pea.エリシターを処理したエンドウ上胚軸由来のRNAから作成されたcDNAライブラリーからエリシター処理により発現が増高する遺伝子候補のcDNAとしてPsDof1が単離された．大腸菌で生産されたGST-PsDof１融合タンパク質はAAAG配列をコアとするDNAに結合することが明らかにされている．本論文ではGST-PsDof１がエリシター応答性遺伝子の一つ，PsCHS1のプロモーター上のAAAGまたはCTTT配列を有する断片に特異的に結合することを明らかにした．更にAAAG配列のエリシター応答性シスエレメントとしての機能を解析するため，AAAG配列を４回繰り返したユニットをCaMV35Ｓの最小プロモーターとCATレポーター遺伝子に連結したキメラ遺伝子を構築し，エンドウプロトプラストにエレクトロポレーション法により導入した．CAT活性を指にプロモーター活性を調べたところ，AAAG配列を有するプロモーターは，エリシター処理により活性化されることが明らかとなった．これらの結果はPsDof１がエリシター応答性防御遺伝子のプロモーター上のAAAG配列に結合し，転写を活性化させる可能性を示唆している

Suitability of Science Experiment Classes for Class Reform

departmental bulletin pape

Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci

A glycosylation island is a genetic region required for glycosylation. The glycosylation island of flagellin in Pseudomonas syringae pv. tabaci 6605 consists of three orfs: orf1, orf2 and orf3. Orf1 and orf2 encode putative glycosyltransferases, and their deletion mutants, Delta orf1 and Delta orf2, exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild-type bacteria and non-glycosylated flagellin from Delta orf1 mutant using aspartic N-peptidase and subsequent HPLC analysis revealed candidate glycosylated amino acids. By generation of site-directed Ser/Ala-substituted mutants, all glycosylated amino acid residues were identified at positions 143, 164, 176, 183, 193 and 201. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis revealed that each glycan was about 540 Da. While all glycosylation-defective mutants retained swimming ability, swarming ability was reduced in the Delta orf1, Delta orf2 and Ser/Ala-substituted mutants. All glycosylation mutants were also found to be impaired in the ability to adhere to a polystyrene surface and in the ability to cause disease in tobacco. Based on the predicted tertiary structure of flagellin, S176 and S183 are expected to be located on most external surface of the flagellum. Thus the effect of Ala-substitution of these serines is stronger than that of other serines. These results suggest that glycosylation of flagellin in P. syringae pv. tabaci 6605 is required for bacterial virulence. It is also possible that glycosylation of flagellin may mask elicitor function of flagellin molecule

Gac two-component system in Pseudomonas syringae pv. tabaci is required for virulence but not for hypersensitive reaction

Pseudomonas syringae pv. tabaci 6605 causes wildfire disease on host tobacco plants. To investigate the regulatory mechanism of the expression of virulence, Gac two-Component system-defective mutants, Delta gacA and Delta gacS, and a double mutant, Delta gacA Delta gacS, were generated. These mutants produced smaller amounts of N-acyl homoserine lactones required for quorum sensing, had lost swarming motility, and had reduced expression of virulence-related hrp genes and the algT gene required for exopolysaccharide production. The ability of the mutants to cause disease symptoms in their host tobacco plant was remarkably reduced, while they retained the ability to induce hypersensitive reaction (HR) in the nonhost plants. These results indicated that the Gac two-component system of P. syringae pv. tabaci 6605 is indispensable for virulence on the host plant, but not for HR induction in the nonhost plants.</p

Elicitin-responsive lectin-like receptor kinase genes in BY-2 cells

The inhibition of elicitor-induced plant defense responses by the protein kinase inhibitors K252a and staurosporine indicates that defense responses require protein phosphorylation. We isolated a cDNA clone encoding Nicotiana tabacum lectin-like receptor protein kinase 1 ( NtlecRK1), an elicitor-responsive gene; in tobacco bright yellow ( BY-2) cells by a differential display method. NtlecRK forms a gene family with at least three members in tobacco. All three NtlecRK genes potentially encode the N-terminal legume lectin domain, transmembrane domain and C-terminal Ser/Thr-type protein kinase domain. Green fluorescent protein ( GFP) fusion showed that the NtlecRK1 protein was located on the plasma membrane. In addition, NtlecRK1 and 3 were responsive to INF1 elicitin and the bacterial elicitor harpin. These results indicate that NtlecRKs are membrane-located protein kinases that are induced during defense responses in BY-2 cells.</p

Modulation of defense signal transduction by flagellin-induced WRKY41 transcription factor in Arabidopsis thaliana

Flagellin, a component of the flagellar filament of Pseudomonas syringae pv. tabaci 6605 (Pta), induces hypersensitive reaction in its non-host Arabidopsis thaliana. We identified the WRKY41 gene, which belongs to a multigene family encoding WRKY plant-specific transcription factors, as one of the flagellin-inducible genes in A. thaliana. Expression of WRKY41 is induced by inoculation with the incompatible pathogen P. syringae pv. tomato DC3000 (Pto) possessing AvrRpt2 and the non-host pathogens Pta within 6-h after inoculation, but not by inoculation with the compatible Pto. Expression of WRKY41 was also induced by inoculation of A. thaliana with an hrp-type three secretion system (T3SS)-defective mutant of Pto, indicating that effectors produced by T3SS in the Pto wild-type suppress the activation of WRKY41. Arabidopsis overexpressing WRKY41 showed enhanced resistance to the Pto wild-type but increased susceptibility to Erwinia carotovora EC1. WRKY41-overexpressing Arabidopsis constitutively expresses the PR5 gene, but suppresses the methyl jasmonate-induced PDF1.2 gene expression. These results demonstrate that WRKY41 may be a key regulator in the cross talk of salicylic acid and jasmonic acid pathways.</p