Research Title: Genetic diversity in some Aegilops species in Jordan revealed using RAPD.

Author: Sameer Masoud, Published Year: 2004

Plant Genetic Resources Newsletter , 139

Faculty: Science

Abstract: Genetic diversity in some Aegilops species in Jordan revealed using RAPD.

Keywords: Aegilops species

Research Title: Performance of some Aegilops species under different water regimes (Research note)

Author: Sameer Masoud, Published Year: 2003

Dirasat, Agriculture Sciences , 30

Faculty: Science

Abstract: Performance of some Aegilops species under different water regimes (Research note)

Keywords: Aegilops species

Research Title: Specificity of different PCR primers for Verticillium dahliae isolated from olive trees in Jordan

Author: Sameer Masoud, Published Year: 2002

Mu’tah Lil-Buhuth wad-Dirasat , 17

Faculty: Science

Abstract: Three pairs of polymerase chain reaction (PCR) primers were compared to amplify characteristic DNA fingerprints of Verticillium dahliae Kleb. (the causal agent of olive vascular wilt disease). The primers include several combinations of the internal transcribed spacer (ITS) regions of nuclear ribosomal RNA (rRNA) genes and one pair from repetitive nuclear DNA sequences. Deoxyoligonucleotide primers specific to V. dahliae were synthesized based on the identified variable nucleotide sequences of the nuclear ITS regions of different Verticillium species that cause vascular wilt diseases. The PCR primers of nuclear repetitive DNA showed variable band intensities for the different isolates of V. dahliae isolated from olive trees in Jordan. This suggests genetic variabilities of the local isolates collected from olive. Primers based on the ITS sequences produced more consistently homogeneous and characteristic fingerprints using purified DNA from V. dahliae isolates. The detection limit of these ITS primers was further improved using nested PCR and to show the high specificity of the ITS primers. The first amplification reaction of nested PCR contained primers from the highly conserved DNA sequences of the 18S and 28S genes that flank the ITS regions. No PCR amplification produced using DNA isolated from different fungi in the same taxonomic class or other classes. These ITS-specific primer pair may be useful in developing diagnostic procedures of Verticillium wilt disease using single or nested PCR. تمت مقارنة ثلثة أزواج من بادئات تفاعل البلمرة التسلسلي(PCR (لتضخيم بصصصمات وراثية خاصة بفطر الفيرتسيليوم دالي المسبب لمرض الذبول الوعائي في الزيتصصون . أشصصتملت البادئات على مزيج من تسلسل المناطق المستنسخة والفاصلة بين جينات بناء الريبوسومات (ITS( وزوج بادئات من تسلسل النيوكليتدات المكررة فصصي الحصصامض النصصووي الريبصصوزي منقصصوص الوكسجين (DNA . (تم تحديد وبناء بادئات من مناطق (ITS (خاصة بالفيرتسيليوم دالي وفصصي المناطق التي تختلف بالتسلسل عن الفطريات الخرى التي تسصبب الصذبول الوعصائي . أنتجصت البادئات من تسلسل النيوكليتدات المكررة وباستعمال أل (PCR (حزم تختلف مصن حيصث الحصدة للعزلت المختلفة من الزيتون في الردن ، ومما يشير لختلفات وراثية بيصن عصزلت الفطصر المختلفة ، بينما أنتجت بادئات أل(ITS (حزم متجانسة لهذه العزلت . كما تم زيادة الحد الدنصصى للكشف عن DNA الفطر باستعمال(PCR (مزدوج وباستعمال بادئات في التفاعل الول مصن جينات (18S (و (28S (المحيطة في منطقة أل (ITS (واستعمال بادئات أل(ITS (في التفاعل الثاني . استعمل هذا الفحص لتأكيد خصوصية البادئات لفطر الفيرتسيليوم دالي وليس لفطريصصات أخرى في نفس الصف التصنيفي أو صفوف أخرى . وسيكون استعمال هذه البادئات في تفاعصصل مفرد أو مزدوج من أل(PCR (مفيدا في تطوير طرق تشخيصية لمرض الذبول الوعصصائي فصصي الزيتون .

Keywords: olive vascular wilt disease

Research Title: Over-expression of cinnamate 4-hydroxylase leads to increased accumulation of acetosyringone in elicited tobacco cell-suspension cultures.

Author: Sameer Masoud, Published Year: 2002

Planta, 214

Faculty: Science

Abstract: Cell-suspension cultures were produced from transgenic tobacco (Nicotiana tabacum L. ) plants harboring a constitutively expressed alfalfa cinnamate 4-hydroxylase (C4H) transgene. Increased levels of C4H enzyme activity in the transgenic cultures were observed only following exposure of the cells to yeast elicitor, although alfalfa C4H transcripts were expressed at a high level from the cauliflower mosaic virus 35S promoter in the absence of elicitation. Increased expression of C4H in elicited cell-suspension cultures had no appreciable effect on the HPLC profiles of soluble phenolic compounds. However, levels of one compound, subsequently identified as 3,5-dimethoxy-4-hydroxy acetophenone (acetosyringone), were strongly elevated in the wall-bound phenolic fraction. The results are discussed in relation to the correlation between C4H activity and the synthesis of 3,5-dimethylated hydroxycinnamic acid derivatives in tobacco.

Keywords: Cell-suspension cultures, cinnamate 4-hydroxylase

Research Title: Altering expression of cinnamic acid 4-hydroxylase in transgenic plants identifies a feedback loop at the entry point into the phenylpropanoid pathway

Author: Sameer Masoud, Published Year: 2000

Plant Physiology , 122

Faculty: Science

Abstract: Pharmacological evidence implicates trans-cinnamic acid as a feedback modulator of the expression and enzymatic activity of the first enzyme in the phenylpropanoid pathway, Lphenylalanine ammonia-lyase (PAL). To test this hypothesis independently of methods that utilize potentially non-specific inhibitors, we generated transgenic tobacco lines with altered activity levels of the second enzyme of the pathway, cinnamic acid 4-hydroxylase (C4H), by sense or antisense expression of an alfalfa C4H cDNA. PAL activity and levels of phenylpropanoid compounds were reduced in leaves and stems of plants in which C4H activity had been genetically down-regulated. However, C4H activity was not reduced in plants in which PAL activity had been down-regulated by gene silencing. In crosses between a tobacco line over-expressing PAL from a bean PAL transgene and a C4H antisense line, progeny populations harboring both the bean PAL sense and C4H antisense transgenes had significantly lower extractable PAL activity than progeny populations harboring the PAL transgene alone. Our data provide genetic evidence for a feedback loop at the entry point into the phenylpropanoid pathway that had previously been inferred from potentially artifactual pharmacological experiments.

Keywords: phenylpropanoid pathway, cinnamic acid 4-hydroxylase

Research Title: The corn inhibitor of activated Hageman factor: purification and properties of two recombinant forms of the protein

Author: Sameer Masoud, Published Year: 1998

Expression and Purification , 13

Faculty: Science

Abstract: A cDNA clone that encodes the 14-kDa bifunctional inhibitor from corn seeds (L. Wenet al., Plant Mol. Biol.18, 813–814, 1992) has been expressed inEscherichia coliafter being incorporated into the pT7 expression vector. This inhibitor protein, referred to as CHFI (for the corn inhibitor of activated Hageman factor) or as the popcorn inhibitor, is an important tool for specific inhibition of human activated Hageman factor (activated forms of coagulation Factor XII) and has been well characterized as isolated from corn seeds. Recombinant CHFI was expressed inE. coliin high levels but was insoluble. We solubilized the expressed protein by sonication in 5 M urea and 1% Triton X-100. Several steps of purification, culminating with reversed-phase HPLC, yielded pure, recombinant corn inhibitor in about 5% yield (about 1 mg per liter of culture). The form with which we have worked most, 7N-CHFI, contains 7 amino acid residues at its N-terminus that are encoded by the expression vector. Physical properties of this recombinant protein indicate it has the expected mass and is properly folded. Functionally, 7N-CHFI is indistinguishable from the inhibitor isolated from corn seeds in its inhibition of porcine trypsin, human β-Factor XIIa, failure to inhibit human plasma kallikrein, and its inhibition of an insect α-amylase. A second recombinant form, (4N-11)-CHFI, which lacks 11 residues from the corn inhibitor's N-terminus, is indistinguishable from 7NCHFI in its pattern of inhibition of the three test proteinases but is inactive against the insect α-amylase. This suggests that the N-terminal region of 7N-CHFI forms at least part of the protein's site of interaction with α-amylase.

Keywords: pT7 expression vector, corn bifunctional inhibitor, Hageman factor

Research Title: Reduced lignin content and altered lignin composition in transgenic tobacco down-regulated in expression of L-phenylalnine ammonia-lyase or cinnamate 4-hydroxylase.

Author: Sameer Masoud, Published Year: 1997

Plant Physiology , 115

Faculty: Science

Abstract: We analyzed lignin content and composition in transgenic tobacco (Nicotiana tabacum) lines altered in the expression of the early phenylpropanoid biosynthetic enzymes Lphenylalanine ammonia-lyase and cinnamate 4-hydroxylase (C4H). The reduction of C4H activity by antisense expression or sense suppression resulted in reduced levels of Klason lignin, accompanied by a decreased syringyl/guaiacyl monomer ratio as determined by pyrolysis gas chromatography/mass spectrometry Similar reduction of lignin levels by down -regulation of L-phenylalanine ammonia-lyase, the enzyme preceding C4H in the central phenylpropanoid pathway, did not result in a decreased syringyl/guaiacyl ratio. Rather, analysis of lignin methoxyl content and pyrolysis suggested an increased syringyl/guaiacyl ratio. One possible explanation of these results is that monolignol biosynthesis from L-phenylalanine might occur by more than one route, even at the early stages of the core phenylpropanoid pathway, prior to the formation of specific monolignol precursors.

Keywords: L-phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, lignin

Research Title: Expression of a corn bifunctional inhibitor of serine proteinase and insect alpha-amylase in transgenic tobacco plants.

Author: Sameer Masoud, Published Year: 1996

Plant Science , 115

Faculty: Science

Abstract: Corn seed endosperm contains a putative defense protein (14K-CI) that inhibits both trypsin-like serine proteinases and insect α-amylases. A cDNA clone encoding 14K-CI under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase polyadenylation region was introduced into tobacco plants by Agrobacterium-mediated transformation. The presence and expression of the chimeric gene in regenerated (R0) and progeny (R1) plants was confirmed by Southern, polymerase chain reaction, and Northern analyses. Protein extracts of selected transformed plants, but not control plants, reacted positively to corn seed 14K-CI antiserum. Comparison of 14K-CI mobilities from transgenic leaves and corn seeds after denaturing polyacrylamide gel electrophoresis indicated that recognition and cleavage of the 14K-CI signal peptide sequence occurred in tobacco leaves. Immunological assays showed that the inhibitor was expressed in amounts up to 0.05% of the total protein in young leaves of R1 plants. Lesser accumulation was generally detected in older leaves. Protein extracts from transgenic plants were more inhibitory than were control plant extracts to bovine trypsin activity. Four tobacco plants with a gene lacking the 14K-CI signal peptide region accumulated 3– 5-fold less inhibitor than did the highest-expressing plant with the full cDNA clone. Use of a double 35S promoter did not enhance 14K-CI accumulation. Post-transcriptional events appear to be a major factor in the low accumulation of 14K-CI in tobacco.

Keywords: transgenic plants, corn inhibitor

Research Title: Constitutive expression of an inducible -1,3-glucanase in alfalfa reduces disease severity caused by the oomycete pathogen Phytopthora megasperma f. sp. medicaginis, but does not reduce disease severity of chitin-containing fungi.

Author: Sameer Masoud, Published Year: 1996

Transgenic Research , 5

Faculty: Science

Abstract: cDNA sequences coding for an acidic glucanase (Aglul) that is expressed in elicited alfalfa cell suspension cultures, and a rice basic chitinase (RCH10) that is induced by elicitor and wounding, were placed into constitutive expression cassettes under control of the cauliflower mosaic virus 35S promoter or 35S enhancer sequences, and introduced in alfalfa plants of the regenerable cultivar Regen SY byAgrobacterium-mediated transformation. Southern and northern blot analysis confirmed stable incorporation and transcription, respectively, of the chimaeric genes in the transgenic plants. Active rice chitinase was expressed in alfalfa leaves, and leaves of plants transformed with theAglul sequence exhibited increased glucanase activity and the appearance of an additional glucanase band on activity gels. A glucanase of similar native electrophoretic mobility was constitutively present in root extracts of non-transformed alfalfa plants, and was induced in pathogen-infected leaves, presumably reflecting the expression pattern of the endogenousAglul gene. Thus, expression of the chimaericAglul transgene increased the amount, and broadened the tissue-type constitutive expression, of theAglul protein compared to control plants. Transgenic alfalfa plants containing a binary vector with both chimaeric genes in tandem expressed each gene to a much lesser extent than transgenic plants containing a single chimaeric gene. Expression of RCH10 in transgenic alfalfa did not appear to affect negatively theRhizobium/alfalfa interaction. Analysis of primary transformants for response to several fungal pathogens of alfalfa indicated statistically significant symptom reduction only in the case ofPhytophthora megasperma f. sp.medicaginis (Pmm), and only in plants overexpressingAglul. Resistance against Pmm segregated with glucanase expression in a cross between transgenic Regen SY and the commercial alfalfa cultivar Apollo.

Keywords: Transgenic alfalfa, Phytophthora megasperma

Research Title: Enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in transgenic tobacco

Author: Sameer Masoud, Published Year: 1994

Bio/technology (Nature Biotechnology), 12

Faculty: Science

Abstract: Plants respond to pathogen attack by the induction of a battery of defenses, suggesting that different protective mechanisms may have complementary roles in the overall expression of disease resistance. We have investigated possible functional interactions between two different hydrolytic enzymes, chitinase and glucanase, by constitutive coexpression in transgenic tobacco of genes encoding the rice RCH10 basic chitinase and the alfalfa AGLU1 acidic glucanase. Hybrid plants were generated by crossing transgenic parental lines exhibiting strong constitutive expression of cauliflower mosaic virus (CaMV) 35S enhancer / RCH10 and CaMV 35S double promoter / AGLU1 gene fusions, respectively. Evaluation of disease development in these hybrids, heterozygous for each transgene, and in homozygous selfed progeny, showed that combination of the two transgenes gave substantially greater protection against the fungal pathogen Cercospora nicotianae, causal agent of frogeye, than either transgene alone. Productive interactions between chitinase and glucanase transgenes in vivo point to combinatorial expression of antimicrobial genes as an effective approach to engineering enhanced crop protection against fungal disease.

Keywords: hydrolytic enzymes, antimicrobial, engineering crop protection