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Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins that inhibit polygalacturonase (PG) enzymes secreted by pathogens to break down plant cell walls during early stage of disease development. Sugar beet ( Beta vulgaris L.) PGIP genes ( BvPGIPs ) have 11 LRR domains as compared to 10 LRRs generally found in other plant species. To determine whether the BvPGIPs have a function in plant defense, genes encoding BvPGIP1 and BvPGIP2 that differ in 8 amino acids were fused with the CaMV 35S constitutive promoter and introduced into Nicotiana benthamiana . Crude PGIP protein extracts from BvPGIP1 transgenic plants significantly inhibited Rhizoctonia solani , Fusarium solani and Botrytis cinerea PGs. BvPGIP2 extract also inhibited PGs from F. solani and B. cinerea but did not inhibit PGs from R . solani . When transgenic BvPGIP plants were bioassayed for resistance, similar results were obtained. This is a first report that documents sugar beet PGIPs with 11 LRRs confer resistance to three different fungal pathogens. [ABSTRACT FROM AUTHOR]

Abstract: Sugar beet root maggot (SBRM, Tetanops myopaeformis von Röder) is a major but poorly understood insect pest of sugar beet (Beta vulgaris L.). The molecular mechanisms underlying plant defense responses are well documented, however, little information is available about complementary mechanisms for insect adaptive responses to overcome host resistance. To date, no studies have been published on SBRM gene expression profiling. Suppressive subtractive hybridization (SSH) generated more than 300 SBRM ESTs differentially expressed in the interaction of the pest with a moderately resistant (F1016) and a susceptible (F1010) sugar beet line. Blast2GO v. 3.2 search indicated that over 40% of the differentially expressed genes had known functions, primarily driven by fruit fly D. melanogaster genes. Expression patterns of 18 selected EST clones were confirmed by RT‐PCR analysis. Gene Ontology (GO) analysis predicted a dominance of metabolic and catalytic genes involved in the interaction of SBRM with its host. SBRM genes functioning during development, regulation, cellular process, signaling and under stress conditions were annotated. SBRM genes that were common or unique in response to resistant or susceptible interactions with the host were identified and their possible roles in insect responses to the host are discussed. [ABSTRACT FROM AUTHOR]

Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins involved in plant defense. Three sugar beet ( Beta vulgaris L.) PGIP genes, BvPGIP1 , BvPGIP2 and BvPGIP3 , were isolated from breeding line F1016 and BvPGIP2 and BvPGIP3 from F1010. Full-length cDNA sequences of the three BvPGIP genes encoded a 382 or 384 amino acid peptide. They shared closest similarity with five PGIP -like sequences found in the sugar beet reference genome (RefBeet 1.2.1) as well as Bv ( FC607 ) PGIP1 and Bv ( FC607 ) PGIP2 from sugar beet breeding line FC607. The predicted structure of BvPGIP1, 2 and 3 peptides was that of Phaseolus vulgaris PvPGIP2 with the exception that the sugar beet peptides were about 40 amino acids longer and encoded an additional LRR repeat. RT-PCR and qPCR analyses demonstrated BvPGIP gene expression in leaves, petioles, hypocotyls and highest expression in roots. Leaf and root BvPGIP transcripts increased in response to insect feeding and mechanical wounding. An agarose radial diffusion assay showed that BvPGIP protein extracts inhibited polygalacturonases (PGs) of Rhizoctonia solani , Fusarium oxysporum f. sp. gladioli and F. oxysporum f. sp. betae with the exception that PGIP extracts from line F1010 did not inhibit R. solani PGs. These findings identify sugar beet PGIP genes that encode LRR domains that are longer than reported for most other species. In addition, the wound up-regulated BvPGIP gene expression and inhibition of fungal PGs by PGIP extracts suggest that like other plant PGIPs, the sugar beet genes may play an active role in defense responses. [ABSTRACT FROM AUTHOR]

Proteinase inhibitors provide a means of engineering plant resistance to insect pests. A Beta vulgaris serine proteinase inhibitor gene (BvSTI) was fused to the constitutive CaMV35S promoter for over-expression in Nicotiana benthamiana plants to study its effect on lepidopteran insect pests. Independently derived BvSTI transgenic tobacco T2 homozygous progeny were shown to have relatively high BvSTI gene transcript levels. BvSTI-specific polyclonal antibodies cross-reacted with the expected 30 kDA recombinant BvSTI protein on Western blots. In gel trypsin inhibitor activity assays revealed a major clear zone that corresponded to the BvSTI proteinase inhibitor that was not detected in the untransformed control plants. BvSTI-transgenic plants were bioassayed for resistance to five lepidopteran insect pests. Spodoptera frugiperda, S. exigua and Manduca sexta larvae fed BvSTI leaves had significant reductions in larval weights as compared to larvae fed on untransformed leaves. In contrast, larval weights increased relative to the controls when Heliothis virescens and Agrotis ipsilon larvae were fed on BvSTI leaves. As the larvae entered the pupal stage, pupal sizes reflected the overall larval weights. Some developmental abnormalities of the pupae and emerging moths were noted. These findings suggest that the sugar beet BvSTI gene may prove useful for effective control of several different lepidopteran insect pests in genetically modified tobacco and other plants. The sugar beet serine proteinase inhibitor may be more effective for insect control because sugar beet is cropped in restricted geographical areas thus limiting the exposure of the insects to sugar beet proteinase inhibitors and build up of non-sensitive midgut proteases. [ABSTRACT FROM AUTHOR]

Expression pattern of a sugar beet serine proteinase inhibitor gene, BvSTI, was characterized in response to mechanical and fall armyworm (FAW; Spodoptera frugiperda J.E. Smith) induced wounding. BvSTI expression was analyzed in three breeding lines moderately resistant to sugar beet root maggot (Tetanops myopaeformis Roder) and in a susceptible line, F1010. Increased mechanical wound induced levels of BvSTI expression were observed in all resistant lines as compared to F1010 during the first 24 h. The most intensive response to wounding was observed in one of the resistant lines, F1016, with a maximum 5- and 2.5-fold increase of BvSTI transcript levels over non-wounded roots and leaves, respectively. In contrast, slight increase of BvSTI transcript levels in leaves and even an initial decrease in roots were observed in F1010. BvSTI transcript accumulation in F1016 and F1010 tissues wounded by FAW showed a similar gene expression pattern, but it was delayed and less intense than the response incited by abiotic wounding. On the protein level, BvSTI specific antibody confirmed increased accumulation of the 30 kDa BvSTI protein in wounded leaves but not in roots of F1016 and F1010. Using trypsin inhibition assays, the activity of BvSTI was confirmed in F1016 roots and leaves and F1010 leaves. In F1010 roots BvSTI activity was completely lacking. We conclude that BvSTI gene expression was wound induced in the insect resistant germplasm suggesting that BvSTI can be used in biotechnological approaches or in breeding programs for improving insect resistance. [ABSTRACT FROM AUTHOR]

Investigates the effect of phytocystatins from rice, oryzacystatin I and II, expressed in alfalfa on the root lesion nematode, Pratylenchus penetrans. Pattern of the gene expression; Reduction in the incidence of root lesions; Potential for the development of the disease resistance.

Two Nicotianaplumbaginifolia cDNA clones, CYP72A2 and npl2, with high sequence similarity to cytochrome P450 monooxygenases were isolated using reverse transcription-polymerase chain reaction. CYP72A2 has an open reading frame of 1524 nucleotides and its deduced 508 amino acid sequence has 45% identity to Catharanthusroseus P450 CYP72A1. npl2 is similar to CYP72A2, except for an 82-nucleotide deletion within its coding region and an internal stop codon. Southern blot analysis indicated that there are at least 3 copies of the CYP72A2 gene and that they are induced at the RNA and protein levels by mechanical wounding, insect chewing (Manducasexta) and cytokinin application. The response to feeding larvae and wounding was systemic. In N. plumbaginifolia plants transformed with a wound-inducible cytokinin biosynthesis gene construct (PI-II-ipt), gene induction in response to wounding and feeding insects occurred sooner and was generally higher than in untransformed plants. The modulation of CYP72A2 expression by cytokinins and the possible role of P450 in plant defense responses are discussed. [ABSTRACT FROM AUTHOR]

Reports that the US Agricultural Research Service (ARS) has received a patent on a modified gene that lets plants put up a stronger and faster defense when insects are eating them. ARS' search for an industry partner for a cooperative research and development agreement; Production of cytokinin, a multipurpose plant hormone.

Since plant root systems capture both water and nutrients essential for the formation of crop yield, there has been renewed biotechnological focus on root system improvement. Although water and nutrient uptake can be facilitated by membrane proteins known as aquaporins and nutrient transporters, respectively, there is a little evidence that root-localised overexpression of these proteins improves plant growth or stress tolerance. Recent work suggests that the major classes of phytohormones are involved not only in regulating aquaporin and nutrient transporter expression and activity, but also in sculpting root system architecture. Root-specific expression of plant and bacterial phytohormone-related genes, using either root-specific or root-inducible promoters or grafting non-transformed plants onto constitutive hormone producing rootstocks, has examined the role of root hormone production in mediating crop stress tolerance. Root-specific traits such as root system architecture, sensing of edaphic stress and root-to-shoot communication can be exploited to improve resource (water and nutrients) capture and plant development under resource-limited conditions. Thus, root system engineering provides new opportunities to maintain sustainable crop production under changing environmental conditions. [ABSTRACT FROM AUTHOR]

Salinity limits crop productivity, in part by decreasing shoot concentrations of the growth-promoting and senescence-delaying hormones cytokinins. Since constitutive cytokinin overproduction may have pleiotropic effects on plant development, two approaches assessed whether specific root-localized transgenic IPT (a key enzyme for cytokinin biosynthesis) gene expression could substantially improve tomato plant growth and yield under salinity: transient root IPT induction (HSP70::IPT) and grafting wild-type (WT) shoots onto a constitutive IPT-expressing rootstock (WT/35S::IPT). Transient root IPT induction increased root, xylem sap, and leaf bioactive cytokinin concentrations 2- to 3-fold without shoot IPT gene expression. Although IPT induction reduced root biomass (by 15%) in control (non-salinized) plants, in salinized plants (100 mM NaCl for 22 d), increased cytokinin concentrations delayed stomatal closure and leaf senescence and almost doubled shoot growth (compared with WT plants), with concomitant increases in the essential nutrient K+ (20%) and decreases in the toxic ion Na+ (by 30%) and abscisic acid (by 20–40%) concentrations in transpiring mature leaves. Similarly, WT/35S::IPT plants (scion/rootstock) grown with 75 mM NaCl for 90 d had higher fruit trans-zeatin concentrations (1.5- to 2-fold) and yielded 30% more than WT/non-transformed plants. Enhancing root cytokinin synthesis modified both shoot hormonal and ionic status, thus ameliorating salinity-induced decreases in growth and yield. [ABSTRACT FROM PUBLISHER]

Cytokinins and cell wall invertase are positive players in regulating fruit sink strength, growth, and yield under salinity as components of the same signalling cascade establishing and developing sink organs.Salinization of water and soil has a negative impact on tomato (Solanum lycopersicum L.) productivity by reducing growth of sink organs and by inducing senescence in source leaves. It has been hypothesized that yield stability implies the maintenance or increase of sink activity in the reproductive structures, thus contributing to the transport of assimilates from the source leaves through changes in sucrolytic enzymes and their regulation by phytohormones. In this study, classical and functional physiological approaches have been integrated to study the influence of metabolic and hormonal factors on tomato fruit sink activity, growth, and yield: (i) exogenous hormones were applied to plants, and (ii) transgenic plants overexpressing the cell wall invertase (cwInv) gene CIN1 in the fruits and de novo cytokinin (CK) biosynthesis gene IPT in the roots were constructed. Although salinity reduces fruit growth, sink activity, and trans-zeatin (tZ) concentrations, it increases the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) during the actively growing period (25 days after anthesis). Indeed, exogenous application of the CK analogue kinetin to salinized actively growing fruits recovered sucrolytic activities (mainly cwInv and sucrose synthase), sink strength, and fruit weight, whereas the ethylene-releasing compound ethephon had a negative effect in equivalent non-stressed fruits. Fruit yield was increased by both the constitutive expression of CIN1 in the fruits (up to 4-fold) or IPT in the root (up to 30%), owing to an increase in the fruit number (lower flower abortion) and in fruit weight. This is possibly related to a recovery of sink activity in reproductive tissues due to both (i) increase in sucrolytic activities (cwInv, sucrose synthase, and vacuolar and cytoplasmic invertases) and tZ concentration, and (ii) a decrease in the ACC levels and the activity of the invertase inhibitor. This study provides new functional evidences about the role of metabolic and hormonal inter-regulation of local sink processes in controlling tomato fruit sink activity, growth, and yield under salinity. [ABSTRACT FROM AUTHOR]

Higher plant metallothioneins are suspected of reducing metal-induced oxidative stress and binding copper and zinc cofactor metals for delivery to important apometalloproteins. A metallothionein-like gene (MT-L2) cDNA clone pCkn16A1 (Accession U35225) was cloned from heat-shock-inducedNicotiana plumbaginifoliacontaining the heat-shock-inducible isopentenyl transferase (ipt) gene (HS-ipt).Iptexpression in plants leads to enhanced cytokinin biosynthesis. In mature leaves of non-transformedN. plumbaginifoliaandNicotiana tabacum, copper stress caused a significant loss of MT-L2 mRNA transcripts. Under non-stressed conditions, HS-iptinduced (N. plumbaginifolia) or light-induced (SSU-ipt) (N. tabacum) plants had higher MT-L2 transcript levels than non-transformed or transformed (GUS) controls. Unlike control plants, copper stress did not reduce MT-L2 mRNA levels in the cytokinin accumulating transgenic plants. Enhanced cytokinin production also led to lower lipid peroxidation compared with controls under non-stressed and copper-stressed conditions. Greater MT-L2 transcript levels and protection against oxidative events prior to or during copper stress could contribute to the observed eight-fold accumulation of copper in mature leaves ofiptexpressing plants compared to non-transformed plants. Expression of this tobacco MT-L2 mRNA may be modulated directly by cytokinin or indirectly as a consequence of cytokinin-mediated antioxidant activity. [ABSTRACT FROM AUTHOR]