Use of Yeast Poly (A) Binding Proteins and Their Genes for Broad Range Protection of Plants Against bacterial, Fungal and Viral Pathogens

Plants that accumulate the yeast polyadenylate binding protein (yPAB) display a range of abnormalities, including a characteristic chlorosis in leaves to a necrosis and pronounced inhibition of growth. The severity of these abnormalities reflects the levels of yeast PAB expression in the transgenic plants. In contrast, no obvious differences are seen in undifferentiated callus cultures that express the same range of yeast PAB. The expression of the yeast PAB1 gene in plants does not affect expression of the plant PAB gene family or alter poly(A) length in the total RNA population. It is proposed that the yeast PAB1 gene or its product interferes with as yet unidentified functions of PABs, which functions are manifest only in differentiated, developed plants. Surprisingly, transgenic plants expressing the yeast PAB1 gene are also observed to have a systemic acquired resistance (SAR) to bacterial, fungal and viral pathogens

Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling

BACKGROUND: The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A) tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A) tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis. RESULTS: By protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (approximately 17%) showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations. CONCLUSION: An extensive protein network was revealed for plant polyadenylation machinery, in which all predicted proteins were found to be connecting to the complex. The gene expression profiles are indicative that specialized sub-complexes may be formed to carry out targeted processing of mRNA in different developmental stages and tissue types. These results offer a roadmap for further functional characterizations of the protein factors, and for building models when testing the genetic contributions of these genes in plant growth and development

Integration of Developmental and Environmental Signals via a Polyadenylation Factor in Arabidopsis

<div><p>The ability to integrate environmental and developmental signals with physiological responses is critical for plant survival. How this integration is done, particularly through posttranscriptional control of gene expression, is poorly understood. Previously, it was found that the 30 kD subunit of Arabidopsis cleavage and polyadenylation specificity factor (AtCPSF30) is a calmodulin-regulated RNA-binding protein. Here we demonstrated that mutant plants (<i>oxt6</i>) deficient in AtCPSF30 possess a novel range of phenotypes – reduced fertility, reduced lateral root formation, and altered sensitivities to oxidative stress and a number of plant hormones (auxin, cytokinin, gibberellic acid, and ACC). While the wild-type AtCPSF30 (C30G) was able to restore normal growth and responses, a mutant AtCPSF30 protein incapable of interacting with calmodulin (C30GM) could only restore wild-type fertility and responses to oxidative stress and ACC. Thus, the interaction with calmodulin is important for part of AtCPSF30 functions in the plant. Global poly(A) site analysis showed that the C30G and C30GM proteins can restore wild-type poly(A) site choice to the <i>oxt6</i> mutant. Genes associated with hormone metabolism and auxin responses are also affected by the <i>oxt6</i> mutation. Moreover, 19 genes that are linked with calmodulin-dependent CPSF30 functions, were identified through genome-wide expression analysis. These data, in conjunction with previous results from the analysis of the <i>oxt6</i> mutant, indicate that the polyadenylation factor AtCPSF30 is a regulatory hub where different signaling cues are transduced, presumably via differential mRNA 3′ end formation or alternative polyadenylation, into specified phenotypic outcomes. Our results suggest a novel function of a polyadenylation factor in environmental and developmental signal integration.</p></div

Global poly(A) site analysis of the four genotypes studied in this report.

<p>Poly(A) site distributions in extended 3′-UTRs were determined on a gene-by-gene basis and used for all possible pair-wise comparisons (wt-<i>oxt6</i>, wt-C30G, etc.) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115779#s2" target="_blank">Methods</a>. Cumulative plots of the difference metric for each pairwise comparison were generated as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115779#pone.0115779-Thomas1" target="_blank">[25]</a> and are shown here. A. Plot of data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115779#pone.0115779-Thomas1" target="_blank">[25]</a>, showing the results of comparisons for replicates from the same line (in this case, the wt; “wt-wt leaf”) the wt and <i>oxt6</i> mutant (“wt-oxt6 leaf”). These curves represent the expected “extremes” of similarity and differences, respectively. B. The comparisons involving the wt, C30G, and G30GM lines were superimposed on those shown in panel A. C. The three comparisons of the <i>oxt6</i> mutant with the other lines were superimposed on those shown in panel A.</p

Characteristics of o<i>xt6</i> flowers.

<p>(A) A wild-type inflorescence showing selected stages of flower development. (B) and (C) flowers from wild-type and oxt6 plants at the +1 stage and +3 stage, respectively. (D) Lengths of the stamens at +1 stage, calculated as a fraction of the lengths of pistils (a value of 1.0 indicates that both organs are the same length). The difference between the wild-type and mutant was significant ate the p<0.05 level by the Student's t-test. (E) Silique phenotypes of the wild-type and <i>oxt6</i> mutant. A typical wild-type inflorescence is shown on the left, and an <i>oxt6</i> inflorescence on the right. The inflorescence in the middle is also from an <i>oxt6</i> plant; in this infloresence, the flowers indicated by the dark arrows were hand-pollinated with pollen from the same <i>oxt6</i> plant.</p

Structures of the transgenes assembled for this study, with an insert showing the wt (A) and mutant CAM-binding domain (B).

<p>A, the genomic DNA is shown at the top, and the two mRNAs beneath. Dark gray boxes indicate the exons present in <i>CPSF30</i>, the smaller of the two transcripts of the <i>OXT6</i> gene; light boxes are additional exons in <i>CPSF30-YT521B</i>, the larger of the two transcripts of <i>OXT6</i>. For brevity, and because the structures are identical for the mutation illustrated at the bottom, only the genomic DNA of the wild-type transgene is shown. The sequences at the bottom of B show the changes used to create the calmodulin-binding mutant; these have been described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115779#pone.0115779-Delaney1" target="_blank">[18]</a>.</p

Lateral root development of the <i>oxt6</i> mutant and complemented plants.

<p>(A) Image of 10-days-old wild-type (wild-type, left) and <i>oxt6</i> (right) seedlings grown on a half MS medium plate. (B) Numbers of lateral roots per primary root per cm in wild-type, <i>oxt6, oxt6</i>::C30G, and <i>oxt6</i>::C30GM lines. Plants were germinated and grown on vertical plates for 10 days, when lateral roots were counted. (C) Primordia in the wild-type and <i>oxt6</i> mutant. Primordia were counted on whole-mount roots of wild-type and <i>oxt6</i> plants 10 days after germination and growth on vertical plates. (D) Numbers of primordia at the different stages of lateral root development (as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115779#pone.0115779-Malamy1" target="_blank">[23]</a>). <i>n</i>>8 plants per column.</p

Results of an analysis of global gene expression in the roots of the wt and <i>oxt6</i> mutant.

<p>Gene expression was measured using the RNA-Seq functionality of CLC Genomics Workbench, and the results analyzed in MAPMAN as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115779#s2" target="_blank">Methods</a>. Bins representing functional groups of genes whose expression is significantly different in the wt and mutant were identified and the p-values that describe the conformance with the hypothesis that the two backgrounds are identical were plotted as shown; for this, the log(10) values for the reciprocal of each p-value was calculated and used in the graph. Bins mentioned in the text are color-coded and described beneath the plot. The full set of bins and p-values for this analysis is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115779#pone.0115779.s002" target="_blank">S2 Table</a>.</p