Use of HRMA Proteins and Their Genes for Broad Range Protection of Plants Against Bacterial, Fungal and Viral Pathogens

The use of an avr gene hrmA to induce systematic acquired resistance in plant cells, plant seeds, plant tissues and plants is disclosed. Also disclosed is the use of low level expression of promoters in combination with the hrmA gene to provide broad-spectrum pathogen resistance in plant cells, plant seeds, plant tissues and plants

Sequence of a putative Vitis vinifera PR-1

Research Note

Effect of Calcium Deficiency on Growth and Leaf Acid Soluble Proteins of Tomato

The effects of temporary Ca (Ca) calcium deficiency lasting 2, 3, 4 or 5 d were investigated on tomato plants at the 6-leaf stage, grown hydroponically under controlled conditions. With 2, 3 or 4 d of Ca deficiency, the dry weight of the tomato leaves, shoots or roots was not different from control. A significant decrease in tomato growth, of up to 70%, appeared on the fifth day. Some visual symptoms were observed on the tomato leaves. The phenomenon concerned was an irreversible mechanism that led to plant death after 12 d, even when Ca was added to the root medium after 2, 3, 4 or 5 d. This is the first report of such a rapid and drastic effect of an essential macronutrient. Moreover, Ca content in leaves during root deprivation showed a decrease in all plants, related to a remobilization toward the apex. Ca could be considered as a partly mobile element: the observation of the youngest leaf limbs by transmission electronic microscopy after 4 d of treatment showed disorganized tissues in a necrotic zone, due to wall impairment related to C deficiency. During temporary Ca deficiency, acid soluble proteins were analyzed in leaves (SDS PAGE electrophoresis / Maldi-TOF). After 4 d of Ca deficiency, protein induction in young leaves was revealed. Three proteins were identified as pathogenesis related proteins (PR-1, PR-3, PR-7) and a threonine deaminase precursor was also found. It was also the first time that pathogenesis related (PR) protein appearance has been shown to be related to Ca deficiency. The PR proteins are generally elicited by pathogen attack. This phenomenon seems to be calcium dependent because other mineral stresses, such as potassium (K) deficiency or sodium (Na) excess, did not reveal acid soluble protein changes. The retranslocation of Ca to young tissue could entail eliciting effects via wall fragments leading to a plant response similar to the response to pathogen attac

Transgenic tobacco plants expressing the Potato virus X open reading frame 3 gene develop specific resistance and necrotic ring symptoms after infection with the homologous virus

Tobacco plants were transformed with the open reading frame 3 gene from Potato virus X (PVX) coding for the p12 protein. Although the transgenic plants exhibited a normal morphological aspect, microscopic examination revealed extensive alterations in leaf tissue structure. After being challenged with PVX, the transgenic plants showed resistance to PVX infection and formation of specific leaf symptoms consisting of concentric rings encircled by necrotic borders. These novel symptoms were accompanied by biochemical changes normally associated with the hypersensitive response (HR) and were absent in noninfected transgenic plants or in PVX-infected nontransgenic plants. No equivalent virus resistance was observed after inoculation with Tobacco mosaic virus or Potato virus Y, suggesting the presence of a specific resistance mechanism. Despite development of HR-like symptoms, systemic acquired resistance was not induced in PVX-infected p12 transgenic plants. No evidence of an RNA-mediated resistance mechanism was found.Fil: Kobayashi, Ken. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Cabral, Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Calamante, Gabriela. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Maldonado, Sara Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Mentaberry, Alejandro Nestor. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Association between the c.*229C>T polymorphism of the topoisomerase IIb binding protein 1 (TopBP1) gene and breast cancer

Topoisomerase IIb binding protein 1 (TopBP1) is involved in cell survival, DNA replication, DNA damage repair and cell cycle checkpoint control. The biological function of TopBP1 and its close relation with BRCA1 prompted us to investigate whether alterations in the TopBP1 gene can influence the risk of breast cancer. The aim of this study was to examine the association between five polymorphisms (rs185903567, rs116645643, rs115160714, rs116195487, and rs112843513) located in the 30UTR region of the TopBP1 gene and breast cancer risk as well as allele-specific gene expression. Five hundred thirty-four breast cancer patients and 556 population controls were genotyped for these SNPs. Allele-specific Top- BP1 mRNA and protein expressions were determined by using real time PCR and western blotting methods, respectively. Only one SNP (rs115160714) showed an association with breast cancer. Compared to homozygous common allele carriers, heterozygous and homozygous for the T variant had significantly increased risk of breast cancer (adjusted odds ratio = 3.81, 95 % confidence interval: 1.63–8.34, p = 0.001). Mean TopBP1 mRNA and protein expression were higher in the individuals with the CT or TT genotype. There was a significant association between the rs115160714 and tumor grade and stage. Most carriers of minor allele had a high grade (G3) tumors classified as T2-T4N1M0. Our study raises a possibility that a genetic variation of TopBP1 may be implicated in the etiology of breast cancer

The miR159-GAMYB pathway: silencing and function of GAMYB homologues amongst diverse plant species

MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression in eukaryotes. In plants, many miRNA families mediate silencing of target genes, which are involved in plant development and plant defence. For my thesis, I have been investigating the miR159-GAMYB pathway, which appears conserved from basal vascular plants to angiosperms. GAMYB transcription factors have been demonstrated to have conserved roles of programmed cell death (PCD) in both the seed aleurone and the anther tapetum in a number of different plant species. However, what the functional role of GAMYB is in vegetative tissues remains unknown. In Arabidopsis, miR159 is critical for proper growth, as its inhibition has a strong negative impact on vegetative growth, due to deregulated GAMYB expression. However, gamyb loss-of-functional mutants display a wild-type phenotype, as their expression is silenced to phenotypically inconsequential levels by miR159 in vegetative tissues. This raises two questions: (1) how is GAMYB so strongly silenced; (2) why is GAMYB strongly and widely transcribed in vegetative tissues for it to be then completely repressed by miR159? These two questions were the focus of my thesis. Firstly, how the Arabidopsis MYB33 and MYB65 are so strongly silenced in the model plant Arabidopsis was investigated. Both genes were predicted to contain a distinctive RNA secondary structure abutting the miR159 binding site, composed of two stem-loop (SL) structures; whereas such SL structures were not predicted to form in other GAMYB-like genes that are targeted less efficiently by miR159 for expression regulation. Functional analysis found that the RNA structure in MYB33 correlated with strong silencing efficacy; introducing mutations to disrupt either SL attenuated miR159 efficacy, while introducing mutations to form an artificial stem-loop structure adjacent to a miRNA-binding site restored strong miR159-mediated silencing. Although how these predicted structures promote miR159-mediated silencing are not determined, we speculate that the stem-loop structures in the vicinity of the miR159 binding site promotes accessibility of the binding site, where if adjacent sequences form strong stem structures, they are less likely to base-pair with binding site nucleotides, maintaining high accessibility of the binding site. Interestingly, the RNA SL structures are predicted to reside in GAMYB-like homologues of numerous angiosperm and gymnosperm plant species, arguing that these structures have been integral in the miR159-GAMYB regulatory relationship over a long period of time. In addition, these structures are not present in the Arabidopsis GAMYB-like homologues that are not transcribed in vegetative tissues, suggesting that selection for such structures only occurs for homologues transcribed in vegetative tissues as to prevent their expression and demarcating them as sensitive targets of miR159. Secondly, to investigate the functional role of the miR159-GAMYB pathway, target MIMIC159 (MIM159) transgenes, which can inhibit endogenous miR159 activity, were expressed in a number of Arabidopsis ecotypes, as well as in tobacco and rice. Inhibiting miR159 in all three plant species resulted in similar phenotypic outcomes, predominantly stunted growth and irregular leaf shape. This implies that the function and expression of the miR159-GAMYB pathway is strongly conserved in distant plant species. This raises several questions: why is GAMYB widely transcribed if its expression is strongly silenced by miR159 throughout the plant to result in little to no phenotypic impact; and why has this been strongly conserved across multiple plant species. When miR159 activity is inhibited in MIM159 tobacco leaves, pathways related to plant defence response are most up-regulated. This included PATHOGENESIS-RELATED PROTEIN (PR) mRNA levels that were 100-1000s fold up-regulated compared to wild type, and correlated with deregulated GAMYB expression. This finding suggests that the miR159-GAMYB pathway is involved in the plant defence response to biotic stress. However, PR expression is not up-regulated in Arabidopsis or rice when miR159 is inhibited, suggesting that despite the conserved nature of the miR159-GAMYB pathway, there are species-specific differences in its function

A study of pathogenesis-related (PR) proteins from pearl millet (Pennisetum glaucum) after infection with the downy mildew pathogen (Sclerospora graminicola)

[no abstract

Characteristic expression of twelve rice PR1 family genes in response to pathogen infection, wounding, and defense-related signal compounds (121/180)

Pathogenesis-related (PR) proteins have been used as markers of plant defense responses, and are classified into 17 families. However, precise information on the majority members in specific PR families is still limited. We were interested in the individual characteristics of rice PR1 family genes, and selected 12 putatively active genes using rice genome databases for expressed genes. All were upregulated upon compatible and/or incompatible rice-blast fungus interactions; three were upregulated in the early infection period and four in the late infection period. Upon compatible rice–bacterial blight interaction, four genes were upregulated, six were not affected, and one was downregulated. These results are in striking contrast to those among 22 ArabidopsisPR1 genes where only one gene was pathogen-inducible. The responses of individual genes to salicylic acid, jasmonic acid, and ethylene induced defense signaling pathways in rice are likely to be different from those in dicot plants. Transcript levels in healthy leaves, roots, and flowers varied according to each gene. Analysis of the partially overlapping expression patterns of rice PR1 genes in healthy tissues and in response to pathogens and other stresses would be useful to understand their possible functions and for use as characteristic markers for defense-related studies in rice

Characterization of the rice pathogen-related protein Rir1a and regulation of the corresponding gene

In rice (Oryza sativa L.), local acquired resistance against Pyricularia oryzae (Cav.), the causal agent of rice blast, can be induced by a preinoculation with the non-host pathogen Pseudomonas syringae pv. syringae. We have cloned a cDNA (Rir1a) and a closely related gene (Rir1b) corresponding to transcripts that accumulate in leaf tissue upon inoculation with P. syringae pv. syringae. The cDNA encodes a putative 107 amino acid protein, Rir1a, that exhibits a putative signal peptide cleavage site in its hydrophobic N-terminal part and a C-terminal part that is relatively rich in glycine and proline. The Rir1b gene contains a Tourist and a Wanderer miniature transposable element in its single intron and encodes a nearly identical protein. Rir1a is similar in sequence (ca. 35% identical and ca. 60% conservatively changed amino acids) to the putative Wir1 family of proteins that are encoded by pathogen-induced transcripts in wheat. Using antibodies raised against a Rir1a-fusion protein we show that Rir1a is secreted from rice protoplasts transiently expressing a 35S::Rir1a construct and that the protein accumulates in the cell wall compartment of rice leaves upon inoculation with P. syringae pv. syringae. Possible roles of Rir1a in pathogen defense are discusse