María Eugenia Zanetti

Cross-Kingdom Comparison of Transcriptomic Adjustments to Low-Oxygen Stress Highlights Conserved and Plant-Specific Responses

High-throughput technology has facilitated genome-scale analyses of transcriptomic adjustments in response to environmental perturbations with an oxygen deprivation component, such as transient hypoxia or anoxia, root waterlogging, or complete submergence. We showed previously that Arabidopsis (Arabidopsis thaliana) seedlings elevate the levels of hundreds of transcripts, including a core group of 49 genes that are prioritized for translation across cell types of both shoots and roots. To recognize low-oxygen responses that are evolutionarily conserved versus species specific, we compared the transcriptomic reconfiguration in 21 organisms from four kingdoms (Plantae, Animalia, Fungi, and Bacteria). Sorting of organism proteomes into clusters of putative orthologs identified broadly conserved responses associated with glycolysis, fermentation, alternative respiration, metabolite transport, reactive oxygen species amelioration, chaperone activity, and ribosome biogenesis. Differentially regulated genes involved in signaling and transcriptional regulation were poorly conserved across kingdoms. Strikingly, nearly half of the induced mRNAs of Arabidopsis seedlings encode proteins of unknown function, of which over 40% had up-regulated orthologs in poplar (Populus trichocarpa), rice (Oryza sativa), or Chlamydomonas reinhardtii. Sixteen HYPOXIA-RESPONSIVE UNKNOWN PROTEIN (HUP) genes, including four that are Arabidopsis specific, were ectopically overexpressed and evaluated for their effect on seedling tolerance to oxygen deprivation. This allowed the identification of HUPs coregulated with genes associated with anaerobic metabolism and other processes that significantly enhance or reduce stress survival when ectopically overexpressed. These findings illuminate both broadly conserved and plant-specific low-oxygen stress responses and confirm that plant-specific HUPs with limited phylogenetic distribution influence low-oxygen stress endurance.

Immunopurification of Polyribosomal Complexes of Arabidopsis for Global Analysis of Gene Expression

Immunoaffinity purification of polyribosomes (polysomes) from crude leaf extracts of Arabidopsis (Arabidopsis thaliana) was achieved with transgenic genotypes that overexpress a translational fusion of a ribosomal protein (RP) with a His6-FLAG dual epitope tag. In plants with a cauliflower mosaic virus 35S:HF-RPL18 transgene immunopurification with anti-FLAG agarose beads yielded 60-Svedberg ribosomal subunits, intact 80-Svedberg monosomes and polysomes. Sucrose density gradient fractionation of the purified complexes demonstrated that the distribution of polysome size was similar in crude cell extracts and the purified complexes. The immunopurified complexes included putative cytosolic RPs of Arabidopsis and ribosome-associated proteins, as well as full-length transcripts of high and low abundance. Whole-genome profiling using long DNA oligonucleotide-based microarrays provided a high level of reproducibility between polysomal mRNA samples immunopurified from two independent biological replicates (r approximately 0.90). Comparison of immunopurified and total cellular RNA samples revealed that for most of the genes, the mRNAs were associated with the epitope-tagged polysomal complexes, with an average relative level of association of 62.06% ± 4.39%. The results demonstrate that the immunopurification of polysomes can be a valuable tool for the quantification of mRNAs present in translation complexes in plant cells. This technology can be extended to evaluation of mRNA populations at the cell- or tissue-specific level by regulation of the tagged RP with distinct promoters.

A C Subunit of the Plant Nuclear Factor NF-Y Required for Rhizobial Infection and Nodule Development Affects Partner Selection in the Common Bean–Rhizobium etli Symbiosis

This study provides evidence that a plant nuclear factor plays a key role in the development of functional nitrogen-fixing nodules and demonstrates that constitutive expression of this gene is sufficient not only to improve nodulation performance of less efficient rhizobium strains but also to enhance the rate of nodule occupancy by strains that are bad competitors in the soil. Legume plants are able to interact symbiotically with soil bacteria to form nitrogen-fixing root nodules. Although specific recognition between rhizobia and legume species has been extensively characterized, plant molecular determinants that govern the preferential colonization by different strains within a single rhizobium species have received little attention. We found that the C subunit of the heterotrimeric nuclear factor NF-Y from common bean (Phaseolus vulgaris) NF-YC1 plays a key role in the improved nodulation seen by more efficient strains of rhizobia. Reduction of NF-YC1 transcript levels by RNA interference (RNAi) in Agrobacterium rhizogenes–induced hairy roots leads to the arrest of nodule development and defects in the infection process with either high or low efficiency strains. Induction of three G2/M transition cell cycle genes in response to rhizobia was impaired or attenuated in NF-YC1 RNAi roots, suggesting that this transcription factor might promote nodule development by activating cortical cell divisions. Furthermore, overexpression of this gene has a positive impact on nodulation efficiency and selection of Rhizobium etli strains that are naturally less efficient and bad competitors. Our findings suggest that this transcription factor might be part of a mechanism that links nodule organogenesis with an early molecular dialogue that selectively discriminates between high- and low-quality symbiotic partners, which holds important implications for optimizing legume performance.

Characterization of an extracellular serine protease of Fusarium eumartii and its action on pathogenesis related proteins

Proteases have been proposed as part of the invasion strategies of some pathogenic fungi. In this work, a serine protease produced by the phytopathogenic fungus Fusarium solani f.sp. eumartii was purified and characterized. Purification of the enzyme was accomplished by gel filtration through a Superose 12 column, followed by hydrophobic interaction chromatography in Phenyl Superose and gel filtration chromatography through Superdex 75. Analysis of the purified enzyme by SDS/PAGE without heat treatment, revealed a single band, which corresponded to the proteolytic activity detected by zymogram. When this protein was subjected to denaturing conditions, two major polypeptides of approximately 30 and 33kDa were revealed. The N-terminal amino acid sequence of one of these polypeptides showed a high similarity with fungal mature serine proteases of the subtilisin family. This protease hydrolysed in vitro, specific polypeptides of potato intercellular washing fluids and cell walls. The protease was also able to degrade pathogenesis-related proteins from the intercellular washing fluids. The role of this serine protease as part of the fungal strategy to colonize potato tuber tissues is discussed.

Selective recruitment of mRNAs and miRNAs to polyribosomes in response to rhizobia infection in Medicago truncatula.

Translation of mRNAs is a key regulatory step that contributes to the coordination and modulation of eukaryotic gene expression during development or adaptation to the environment. mRNA stability or translatability can be regulated by the action of small regulatory RNAs (sRNAs), which control diverse biological processes. Under low nitrogen conditions, leguminous plants associate with soil bacteria and develop a new organ specialized in nitrogen fixation: the nodule. To gain insight into the translational regulation of mRNAs during nodule formation, the association of mRNAs and sRNAs to polysomes was characterized in roots of the model legume Medicago truncatula during the symbiotic interaction with Sinorhizobium meliloti. Quantitative comparison of steady-state and polysomal mRNAs for 15 genes involved in nodulation identified a group of transcripts with slight or no change in total cellular abundance that were significantly upregulated at the level of association with polysomes in response to rhizobia. This group included mRNAs encoding receptors like kinases required either for nodule organogenesis, bacterial infection or both, and transcripts encoding GRAS and NF-Y transcription factors (TFs). Quantitative analysis of sRNAs in total and polysomal RNA samples revealed that mature microRNAs (miRNAs) were associated with the translational machinery, notably, miR169 and miR172, which target the NF-YA/HAP2 and AP2 TFs, respectively. Upon inoculation, levels of miR169 pronouncedly decreased in polysomal complexes, concomitant with the increased accumulation of the NF-YA/HAP2 protein. These results indicate that both mRNAs and miRNAs are subject to differential recruitment to polysomes, and expose the importance of selective mRNA translation during root nodule symbiosis.

A Small GTPase of the Rab Family Is Required for Root Hair Formation and Preinfection Stages of the Common Bean–Rhizobium Symbiotic Association

Legume plants are able to establish a symbiotic relationship with soil bacteria from the genus Rhizobium, leading to the formation of nitrogen-fixing root nodules. Successful nodulation requires both the formation of infection threads (ITs) in the root epidermis and the activation of cell division in the cortex to form the nodule primordium. This study describes the characterization of RabA2, a common bean (Phaseolus vulgaris) cDNA previously isolated as differentially expressed in root hairs infected with Rhizobium etli, which encodes a protein highly similar to small GTPases of the RabA2 subfamily. This gene is expressed in roots, particularly in root hairs, where the protein was found to be associated with vesicles that move along the cell. The role of this gene during nodulation has been studied in common bean transgenic roots using a reverse genetic approach. Examination of root morphology in RabA2 RNA interference (RNAi) plants revealed that the number and length of the root hairs were severely reduced in these plants. Upon inoculation with R. etli, nodulation was completely impaired and no induction of early nodulation genes (ENODs), such as ERN1, ENOD40, and Hap5, was detected in silenced hairy roots. Moreover, RabA2 RNAi plants failed to induce root hair deformation and to initiate ITs, indicating that morphological changes that precede bacterial infection are compromised in these plants. We propose that RabA2 acts in polar growth of root hairs and is required for reorientation of the root hair growth axis during bacterial infection.

Host Genes Involved in Nodulation Preference in Common Bean (Phaseolus vulgaris)-Rhizobium etli Symbiosis Revealed by Suppressive Subtractive Hybridization

Common bean cultivars are nodulated preferentially by Rhizobium etli lineages from the same center of host diversification. Nodulation was found to be earlier and numerous in bean plants inoculated with the cognate strain. We predicted that analysis of transcripts at early stages of the interaction between host and rhizobium would identify plant genes that are most likely to be involved in this preferential nodulation. Therefore, we applied a suppressive subtractive hybridization approach in which cDNA from a Mesoamerican cultivar inoculated with either the more- or less-efficient strain of R. etli was used as the driver and the tester, respectively. Forty-one independent tentative consensus sequences (TCs) were obtained and classified into different functional categories. Of 11 selected TCs, 9 were confirmed by quantitative reverse-transcriptase polymerase chain reaction. Two genes show high homology to previously characterized plant receptors. Two other upregulated genes encode for Rab11, a member of the small GTP-binding protein family, and HAP5, a subunit of the heterotrimeric CCAAT-transcription factor. Interestingly, one of the TCs encodes for an isoflavone reductase, which may lead to earlier Nod factor production by specific strains of rhizobia. The transcript abundance of selected cDNAs also was found to be higher in mature nodules of the more efficient interaction. Small or no differences were observed when an Andean bean cultivar was inoculated with a cognate strain, suggesting involvement of these genes in the strain-specific response. The potential role of these genes in the early preferential symbiotic interaction is discussed.

A Nuclear Factor Y Interacting Protein of the GRAS Family Is Required for Nodule Organogenesis, Infection Thread Progression, and Lateral Root Growth

Initiation and development of nodules during legume-rhizobia symbiosis and lateral root growth are modulated by two interacting transcription factors. A C subunit of the heterotrimeric nuclear factor Y (NF-YC1) was shown to play a key role in nodule organogenesis and bacterial infection during the nitrogen fixing symbiosis established between common bean (Phaseolus vulgaris) and Rhizobium etli. To identify other proteins involved in this process, we used the yeast (Saccharomyces cerevisiae) two-hybrid system to screen for NF-YC1-interacting proteins. One of the positive clones encodes a member of the Phytochrome A Signal Transduction1 subfamily of GRAS (for Gibberellic Acid-Insensitive (GAI), Repressor of GAI, and Scarecrow) transcription factors. The protein, named Scarecrow-like13 Involved in Nodulation (SIN1), localizes both to the nucleus and the cytoplasm, but in transgenic Nicotiana benthamiana cells, bimolecular fluorescence complementation suggested that the interaction with NF-YC1 takes place predominantly in the nucleus. SIN1 is expressed in aerial and root tissues, with higher levels in roots and nodules. Posttranscriptional gene silencing of SIN1 using RNA interference (RNAi) showed that the product of this gene is involved in lateral root elongation. However, root cell organization, density of lateral roots, and the length of root hairs were not affected by SIN1 RNAi. In addition, the expression of the RNAi of SIN1 led to a marked reduction in the number and size of nodules formed upon inoculation with R. etli and affected the progression of infection threads toward the nodule primordia. Expression of NF-YA1 and the G2/M transition cell cycle genes CYCLIN B and Cell Division Cycle2 was reduced in SIN1 RNAi roots. These data suggest that SIN1 plays a role in lateral root elongation and the establishment of root symbiosis in common bean.

A Phylogenetically Conserved Group of Nuclear Factor-Y Transcription Factors Interact to Control Nodulation in Legumes

A phylogenetically conserved group of transicription factors form trimers to regulate root nodule development and early gene expression during the legume-Rhizobium symbiosis. The endosymbiotic association between legumes and soil bacteria called rhizobia leads to the formation of a new root-derived organ called the nodule in which differentiated bacteria convert atmospheric nitrogen into a form that can be assimilated by the host plant. Successful root infection by rhizobia and nodule organogenesis require the activation of symbiotic genes that are controlled by a set of transcription factors (TFs). We recently identified Medicago truncatula nuclear factor-YA1 (MtNF-YA1) and MtNF-YA2 as two M. truncatula TFs playing a central role during key steps of the Sinorhizobium meliloti-M. truncatula symbiotic interaction. NF-YA TFs interact with NF-YB and NF-YC subunits to regulate target genes containing the CCAAT box consensus sequence. In this study, using a yeast two-hybrid screen approach, we identified the NF-YB and NF-YC subunits able to interact with MtNF-YA1 and MtNF-YA2. In yeast (Saccharomyces cerevisiae) and in planta, we further demonstrated by both coimmunoprecipitation and bimolecular fluorescence complementation that these NF-YA, -B, and -C subunits interact and form a stable NF-Y heterotrimeric complex. Reverse genetic and chromatin immunoprecipitation-PCR approaches revealed the importance of these newly identified NF-YB and NF-YC subunits for rhizobial symbiosis and binding to the promoter of MtERN1 (for Ethylene Responsive factor required for Nodulation), a direct target gene of MtNF-YA1 and MtNF-YA2. Finally, we verified that a similar trimer is formed in planta by the common bean (Phaseolus vulgaris) NF-Y subunits, revealing the existence of evolutionary conserved NF-Y protein complexes to control nodulation in leguminous plants. This sheds light on the process whereby an ancient heterotrimeric TF mainly controlling cell division in animals has acquired specialized functions in plants.

Translating Ribosome Affinity Purification (TRAP) followed by RNA sequencing technology (TRAP-SEQ) for quantitative assessment of plant translatomes.

Translating Ribosome Affinity Purification (TRAP) is a technology to isolate the population of mRNAs associated with at least one 80S ribosome, referred as the translatome. TRAP is based on the expression of an epitope-tagged version of a ribosomal protein and the affinity purification of ribosomes and associated mRNAs using antibodies conjugated to agarose beads. Quantitative assessment of the translatome is achieved by direct RNA sequencing (RNA-SEQ), which provides accurate quantitation of ribosome-associated mRNAs and reveals alternatively spliced isoforms. Here we present a detailed procedure for TRAP, as well as a guide for preparation of RNA-SEQ libraries (TRAP-SEQ) and a primary data analysis. This methodology enables the study of translational dynamic by assessing rapid changes in translatomes, at organ or cell-type level, during development or in response to endogenous or exogenous stimuli.