Murilo S. Alves

Plant bZIP Transcription Factors Responsive to Pathogens: A Review

Transcription factors of the basic leucine zipper (bZIP) family control important processes in all eukaryotes. In plants, bZIPs are master regulators of many central developmental and physiological processes, including morphogenesis, seed formation, abiotic and biotic stress responses. Modulation of the expression patterns of bZIP genes and changes in their activity often contribute to the activation of various signaling pathways and regulatory networks of different physiological processes. However, most advances in the study of plant bZIP transcription factors are related to their involvement in abiotic stress and development. In contrast, there are few examples of functional research with regard to biotic stress, particularly in the defense against pathogens. In this review, we summarize the recent progress revealing the role of bZIP transcription factors in the biotic stress responses of several plant species, from Arabidopsis to cotton. Moreover, we summarize the interacting partners of bZIP proteins in molecular responses during pathogen attack and the key components of the signal transduction pathways with which they physically interact during plant defense responses. Lastly, we focus on the recent advances regarding research on the functional role of bZIPs in major agricultural cultivars and examine the studies performed in this field.

Transcription Factor Functional Protein-Protein Interactions in Plant Defense Responses

Responses to biotic stress in plants lead to dramatic reprogramming of gene expression, favoring stress responses at the expense of normal cellular functions. Transcription factors are master regulators of gene expression at the transcriptional level, and controlling the activity of these factors alters the transcriptome of the plant, leading to metabolic and phenotypic changes in response to stress. The functional analysis of interactions between transcription factors and other proteins is very important for elucidating the role of these transcriptional regulators in different signaling cascades. In this review, we present an overview of protein-protein interactions for the six major families of transcription factors involved in plant defense: basic leucine zipper containing domain proteins (bZIP), amino-acid sequence WRKYGQK (WRKY), myelocytomatosis related proteins (MYC), myeloblastosis related proteins (MYB), APETALA2/ ETHYLENE-RESPONSIVE ELEMENT BINDING FACTORS (AP2/EREBP) and no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF), and cup-shaped cotyledon (CUC) (NAC). We describe the interaction partners of these transcription factors as molecular responses during pathogen attack and the key components of signal transduction pathways that take place during plant defense responses. These interactions determine the activation or repression of response pathways and are crucial to understanding the regulatory networks that modulate plant defense responses.

A novel transcription factor, ERD15 (Early Responsive to Dehydration 15), connects endoplasmic reticulum stress with an osmotic stress-induced cell death signal.

As in all other eukaryotic organisms, endoplasmic reticulum (ER) stress triggers the evolutionarily conserved unfolded protein response in soybean, but it also communicates with other adaptive signaling responses, such as osmotic stress-induced and ER stress-induced programmed cell death. These two signaling pathways converge at the level of gene transcription to activate an integrated cascade that is mediated by N-rich proteins (NRPs). Here, we describe a novel transcription factor, GmERD15 (Glycine max Early Responsive to Dehydration 15), which is induced by ER stress and osmotic stress to activate the expression of NRP genes. GmERD15 was isolated because of its capacity to stably associate with the NRP-B promoter in yeast. It specifically binds to a 187-bp fragment of the NRP-B promoter in vitro and activates the transcription of a reporter gene in yeast. Furthermore, GmERD15 was found in both the cytoplasm and the nucleus, and a ChIP assay revealed that it binds to the NRP-B promoter in vivo. Expression of GmERD15 in soybean protoplasts activated the NRP-B promoter and induced expression of the NRP-B gene. Collectively, these results support the interpretation that GmERD15 functions as an upstream component of stress-induced NRP-B-mediated signaling to connect stress in the ER to an osmotic stress-induced cell death signal.

EARLY RESPONSIVE to DEHYDRATION 15, a new transcription factor that integrates stress signaling pathways

The Early Responsive to Dehydration (ERD) genes are defined as those genes that are rapidly activated during drought stress. The encoded proteins show a great structural and functional diversity, with a particular class of proteins acting as connectors of stress response pathways. Recent studies have shown that ERD15 proteins from different species of plants operate in cross-talk among different response pathways. In this mini-review, we show the recent progress on the functional role of this diverse family of proteins and demonstrate that a soybean ERD15 homolog can act as a connector in stress response pathways that trigger a programmed cell death signal.

Interaction between the New World begomovirus Euphorbia yellow mosaic virus and its associated alphasatellite: effects on infection and transmission by the whitefly Bemisia tabaci

The majority of Old World monopartite begomoviruses (family Geminiviridae) are associated with satellite DNAs. Alphasatellites are capable of autonomous replication, but depend on the helper virus for movement, encapsidation and transmission by the insect vector. Recently, Euphorbia yellow mosaic alphasatellite (EuYMA) was found in association with Euphorbia yellow mosaic virus (EuYMV) infecting Euphorbia heterophylla plants in Brazil. The geographical range of EuYMA was assessed in a representative sampling of E. heterophylla plants collected in several states of Brazil from 2009 to 2014. Infectious clones were generated and used to assess the phenotype of viral infection in the presence or absence of the alphasatellite in tomato, E. heterophylla, Nicotiana benthamiana, Arabidopsis thaliana and Crotalaria juncea. Phenotypic differences of EuYMV infection in the presence or absence of EuYMA were observed in A. thaliana, N. benthamiana and E. heterophylla. Symptoms were more severe when EuYMV was inoculated in combination with EuYMA in N. benthamiana and E. heterophylla, and the presence of the alphasatellite was determinant for symptom development in A. thaliana. Quantification of EuYMV and EuYMA indicated that EuYMA affects the accumulation of EuYMV during infection on a host-dependent basis. Transmission assays indicated that EuYMA negatively affects the transmission of EuYMV by Bemisia tabaci MEAM1. Together, these results indicate that EuYMA is capable of modulating symptoms, viral accumulation and whitefly transmission of EuYMV, potentially interfering with virus dissemination in the field.

Differential expression of four soybean bZIP genes during Phakopsora pachyrhizi infection

Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, is one of most important diseases in the soybean (Glycine max (L.) Merr.) agribusiness. The identification and characterization of genes related to plant defense responses to fungal infection are essential to develop ASR-resistant plants. In this work, we describe four soybean genes, GmbZIP62, GmbZIP105, GmbZIPE1, and GmbZIPE2, which encode transcription factors containing a basic leucine zipper (bZIP) domain from two divergent classes, and that are responsive to P. pachyrhizi infection. Molecular phylogenetic analyses demonstrated that these genes encode proteins similar to bZIP factors responsive to pathogens. Yeast transactivation assays showed that only GmbZIP62 has strong transactivation activity in yeast. In addition, three of the bZIP transcription factors analyzed were also differentially expressed by plant defense hormones, and all were differentially expressed by fungal attack, indicating that these proteins might participate in response to ASR infection. The results suggested that these bZIP proteins are part of the plant defense response to P. pachyrhizi infection, by regulating the gene expression related to ASR infection responses. These bZIP genes are potential targets to obtain new soybean genotypes resistant to ASR.

Expressão gênica do colágeno em ferida cutânea de equinos tratada com plasma rico em plaquetas

ABSTRACT.- Souza M.V., Pinto J.O., Costa M.B.M., Alves M.S., Silva M.O., Martinho K.O. & Fiet -to L.G. 2014. [ Collagen gene expression in skin wound of horses treated with platelet-rich plasma . ] Expressao genica do colageno em ferida cutânea de equinos tratada com plasma rico em plaquetas . Pesquisa Veterinaria Brasileira 34(3):233-240 . Departamento de Veterinaria, Universidade Federal de Vicosa, Campus Universitario s/n, Vicosa, MG 36570-000, Brazil. E-mail: msouza@ufv.brPlatelet-rich plasma (PRP) is a product derived from total blood centrifugation, whose use is focused on improving the healing of different tissues, as a result of the growth factors it contains. However, the clinical benefits of this therapy have not been fully established. The objective of this study was to evaluate type I and III collagen gene expression during different phases of the healing process of PRP-treated skin. Eight healthy crossbred gel-dings, aged 16 and 17 years (16.37±0.52) were used. Three quadrangular-shaped lesions (6.25cm

Functional Diversity of Early Responsive to Dehydration (ERD) Genes in Soybean

In many regions of the world, agriculture is the primary consumer of water. As the world population increases, and arid regions become more abundant, water will become an in‐ creasingly scarce resource [1]. In 2011, the world’s soybean crops produced 263.7 million tons from an area of 103,5 million hectares [2]. This global production required an input of 0,2 to 0,25 inch of water per acre per day during peak demand, which represents a major problem for the producer countries [3]. In Brazil, the second largest soybean producer in the world, there was a 7% reduction in soybean production in 2011/2012 compared to the previ‐ ous season. This yield loss can be attributed to drought in the soybean-growing regions of the country, which in turn resulted in increased use of irrigation water in an attempt to min‐ imize yield losses [4].

Selection of reference genes for quantitative PCR analysis in Citrus aurantifolia during phytoplasma infection

Quantifying gene expression is essential in most functional genomics experiments. For quantitative PCR (qPCR) assays, reproducible results are dependent on the correct choice of the reference genes for data normalization. To date, screenings for candidate reference genes suitable for expression studies on plant-phytoplasma interactions in plants have not been reported. In the present study, we analyzed the expression patterns of 14 genes in midrib samples of C. aurantifolia plants infected with a ‘Candidatus Phytoplasma aurantifolia’ strain. Using GeNormPlus, NormFinder and BestKeeper algorithms, as well as testing relative expression by REST2009 software, the expression stability of several “classical” reference genes, such as GAPDH, CYCLOPHILIN and 18S rRNA, and of newly identified candidates, was assessed. Our results showed similar performance among GeNormPlus, NormFinder and BestKeeper in evaluating the suitability of reference genes, with few differences among the top five genes. Furthermore, our data showed that some of the widely used reference genes for relative expression normalization in plants, including citrus lineages, were not the most stably expressed transcripts. In conclusion, we provide a list of validated reference genes and their relative primer sequences, usable to conduct reliable qPCR experiments in C. aurantifolia during phytoplasma infection.

Differential expression and phytohormone unbalance in Citrus aurantifolia plants during “sudden decline of lime”, a new phytoplasma disease of citrus

For decades, the production of acid lime (Citrus aurantifolia) in Oman has been affected by diseases caused by phytoplasmas, notably witches’ broom disease of lime (WBDL). In recent years, a new phytoplasma strain in Oman has been observed that promotes the sudden decline of lime (SDL). The molecular mechanisms behind its pathogenicity and mode of interaction with citrus host plants is still completely unknown. In this study, we evaluated the differential expression of genes in symptomatic and asymptomatic lime trees in Oman using a real-time quantitative PCR assay. Among 27 regulatory and biosynthesis-related genes tested in Citrus aurantifolia plants during phytoplasma infection, we verified the presence of 14 responsive genes in plants showing SDL symptoms, revealing a specific set of SDL-responsive genes. Quantification data of endogenous 3-indoleacetic acid and jasmonic acid show an unbalanced hormonal content in symptomatic lime trees, corroborating the gene expression data. The identification of regulatory genes differentially expressed in plant-phytoplasma interactions during SDL will help to elucidate the mechanisms possibly involved in defense responses, development and death-triggered signals in infected citrus plants.