Insect pests and pathogens (fungi, bacteria and viruses) are responsible for severe crop losses. Insects feed directly on the plant tissues, while the pathogens lead to damage or death of the plant. Plants have evolved a certain degree of resistance through the production of defence compounds, which may be aproteic, e.g. antibiotics, alkaloids, terpenes, cyanogenic glucosides or proteic, e.g. chitinases, b-1,3-glucanases, lectins, arcelins, vicilins, systemins and enzyme inhibitors. The enzyme inhibitors impede digestion through their action on insect gut digestive a-amylases and proteinases, which play a key role in the digestion of plant starch and proteins. The natural defences of crop plants may be improved through the use of transgenic technology. Current research in the area focuses particularly on weevils as these are highly dependent on starch for their energy supply. Six dierent a-amylase inhibitor classes, lectin-like, knottin-like, cereal-type, Kunitz-like, c-purothionin-like and thaumatin-like could be used in pest control. These classes of inhibitors show remarkable structural variety leading to dierent modes of inhibition and dierent speci®city pro®les against diverse a-amylases. Speci®city of inhibition is an important issue as the introduced inhibitor must not adversely aect the plant's own a-amylases, nor the nutritional value of the crop. Of particular interest are some bifunctional inhibitors with additional favourable properties, such as proteinase inhibitory activity or chitinase activity. The area has bene®ted from the recent determination of many structures of a-amylases, inhibitors and complexes. These structures highlight the remarkable variety in structural modes of a-amylase inhibition. The continuing discovery of new classes of a-amylase inhibitor ensures that exciting discoveries remain to be made. In this review, we summarize existing knowledge of insect a-amylases, plant a-amylase inhibitors and their interaction. Positive results recently obtained for transgenic plants and future prospects in the area are reviewed.Keywords: a-amylase inhibitors; knottin-like; lectin-like; thaumatin-like; Kunitz; cereal-type; bean weevils; bifunctional inhibitors.Insect pests and pathogens such as fungi, bacteria and viruses are together, responsible for severe crop losses. Worldwide, losses in agricultural production due to pest attack are around 37%, with small-scale farmers hardest hit [1]. Starchy leguminous seeds are an important staple food and a source of dietary protein in many countries. These seeds are rich in protein, carbohydrate and lipid and therefore suffer extensive predation by bruchids (weevils) and other pests. The larvae of the weevil burrow into the seedpods and seeds and the insects usually continue to multiply during seed storage. The damage causes extensive losses, especially if the seeds are stored for long periods.In general, plants contain a certain degree of resistance against insect predation, which is re¯ected in the limited number of insects capable of feeding on a gi...

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Accuracy in quantitative real-time polymerase chain reaction (qPCR) requires the use of stable endogenous controls. Normalization with multiple reference genes is the gold standard, but their identification is a laborious task, especially in species with limited sequence information. Coffee (Coffea ssp.) is an important agricultural commodity and, due to its economic relevance, is the subject of increasing research in genetics and biotechnology, in which gene expression analysis is one of the most important fields. Notwithstanding, relatively few works have focused on the analysis of gene expression in coffee. Moreover, most of these works have used less accurate techniques such as northern blot assays instead of more accurate techniques (e.g., qPCR) that have already been extensively used in other plant species. Aiming to boost the use of qPCR in studies of gene expression in coffee, we uncovered reference genes to be used in a number of different experimental conditions. Using two distinct algorithms implemented by geNorm and Norm Finder, we evaluated a total of eight candidate reference genes (psaB, PP2A, AP47, S24, GAPDH, rpl39, UBQ10, and UBI9) in four different experimental sets (control versus drought-stressed leaves, control versus droughtstressed roots, leaves of three different coffee cultivars, and four different coffee organs). The most suitable combination of reference genes was indicated in each experimental set for use as internal control for reliable qPCR data normalization. This study also provides useful guidelines for reference gene selection for researchers working with coffee plant samples under conditions other than those tested here.

Drought episodes decrease plant growth and productivity, which in turn cause high economic losses. Plants naturally sense and respond to water stress by activating specific signalling pathways leading to physiological and developmental adaptations. Genetically engineering genes that belong to these pathways might improve the drought tolerance of plants. The abscisic acid (ABA)-responsive element binding protein 1/ABRE binding factor (AREB1/ABF2) is a key positive regulator of the drought stress response. We investigated whether the CRISPR activation (CRISPRa) system that targets AREB1 might contribute to improve drought stress tolerance in Arabidopsis. Arabidopsis histone acetyltransferase 1 (AtHAT1) promotes gene expression activation by switching chromatin to a relaxed state. Stable transgenic plants expressing chimeric dCas9 HAT were first generated. Then, we showed that the CRISPRa dCas9 HAT mechanism increased the promoter activity controlling the β-glucuronidase ( GUS ) reporter gene. To activate the endogenous promoter of AREB1 , the CRISPRa dCas9 HAT system was set up, and resultant plants showed a dwarf phenotype. Our qRT-PCR experiments indicated that both AREB1 and RD29A , a gene positively regulated by AREB1, exhibited higher gene expression than the control plants. The plants generated here showed higher chlorophyll content and faster stomatal aperture under water deficit, in addition to a better survival rate after drought stress. Altogether, we report that CRISPRa dCas9 HAT is a valuable biotechnological tool to improve drought stress tolerance through the positive regulation of AREB1.

The seeds of plants are rich stores of proteins, carbohydrates, and lipids and are therefore used by heterotrophs as valuable food sources. Humans use seeds as a major food source and have learned, through agricultural practice, how to increase the levels and the quality of their components. They have also learned how to deal with the multiplicity of toxic or antinutritional compounds present in seeds. It is believed that these seeds, most of which are not essential for the establishment of the new plant following germination, contribute to the protection and defense of seeds against pathogens and predators. However, insects, fungi, and bacteria have also learned how to cope with detrimental compounds in order to take advantage of the high nutritional value of seeds.Coleopteran insects of the family Bruchidae, the seed weevils, have been associated with the seeds of leguminous plants through co-evolutionary processes. These processes have permitted the weevils to thrive on seeds full of toxic compounds, in contrast to the majority of the other potential aggressors, which are incapable of dealing with them. The association between bruchids and legume seeds is highly specific with only seeds of a very few species being attacked by any one insect species.Among our food sources, plants of the legume family contribute some of the most important protein-rich seeds. The common bean (Phaseolus vulgaris), native of the New World and the cowpea (Vigna unguiculata), which originated in Africa, are heavily attacked by bruchids, both in the field and in storage. Infestations are commonly so heavy that the seeds are unsuitable for use as food, feed, or planting.Control of bruchid infestation is done by treating stored seeds with methyl bromide, carbon disulfide, and several other chemicals. These are considered environmentally undesirable and are too expensive for subsistence farmers. To increase the insect resistance of cultivated varieties plant breeders are interested in understanding resistance mechanisms that operate in wild varieties or why certain bruchids attack one cultivated species but not another.Both the common bean and cowpea are endowed with compounds called general defensive compounds that protect their seeds against widely different herbivores. Among these are the tannins, cyanogenic glucosides, non-protein amino acids, and proteins such as protease and amylase inhibitors, lectins, chitinases, ␤-1,3-glucanases. These defensive compounds are ineffective against the host-specific bruchids, Callosobruchus maculatus and Zabrotes subfasciatus, which attack cowpea and common bean, respectively. Host-specific defenses are rare and are generally found in populations in the centers of dispersion of the particular plant species. Landraces of cowpea and common bean that produce seeds resistant to their associated bruchids have been discovered respectively in West Africa and Mexico. The biochemical basis of the resistance of cowpea and common bean seeds to C. maculatus and Z. subfasciatus, respectively, is the focus of t...

Storage proteins perform essential roles in plant survival, acting as molecular reserves important for plant growth and maintenance, as well as being involved in defense mechanisms by virtue of their properties as insecticidal and antimicrobial proteins. These proteins accumulate in storage vacuoles inside plant cells, and, in response to determined signals, they may be used by the different plant tissues in response to pathogen attack. To shed some light on these remarkable proteins with dual functions, storage proteins found in germinative tissues, such as seeds and kernels, and in vegetative tissues, such as tubercles and leaves, are extensively discussed here, along with the related mechanisms of protein expression. Among these proteins, we focus on 2S albumins, Kunitz proteinase inhibitors, plant lectins, glycine-rich proteins, vicilins, patatins, tarins, and ocatins. Finally, the potential use of these molecules in development of drugs to combat human and plant pathogens, contributing to the development of new biotechnology-based medications and products for agribusiness, is also presented.

Over the years, several studies have been performed to analyse plant–pathogen interactions. Recently, functional genomic strategies, including proteomics and transcriptomics, have contributed to the effort of defining gene and protein function and expression profiles. Using these ‘omic’ approaches, pathogenicity‐ and defence‐related genes and proteins expressed during phytopathogen infections have been identified and enormous datasets have been accumulated. However, the understanding of molecular plant–pathogen interactions is still an intriguing area of investigation. Proteomics has dramatically evolved in the pursuit of large‐scale functional assignment of candidate proteins and, by using this approach, several proteins expressed during phytopathogenic interactions have been identified. In this review, we highlight the proteins expressed during plant–virus, plant–bacterium, plant–fungus and plant–nematode interactions reported in proteomic studies, and discuss these findings considering the advantages and limitations of current proteomic tools.

Summary Micro RNA s (mi RNA s) modulate the abundance and spatial–temporal accumulation of target mRNA s and indirectly regulate several plant processes. Transcriptional regulation of the genes encoding mi RNA s ( MIR genes) can be activated by numerous transcription factors, which themselves are regulated by other mi RNA s. Fine‐tuning of MIR genes or mi RNA s is a powerful biotechnological strategy to improve tolerance to abiotic or biotic stresses in crops of economic importance. Current approaches for mi RNA fine‐tuning are based on the down‐ or up‐regulation of MIR gene transcription and the use of genetic engineering tools to manipulate the final concentration of these mi RNA s in the cytoplasm. Transgenesis, cisgenesis, intragenesis, artificial MIR genes, endogenous and artificial target mimicry, MIR genes editing using Meganucleases, ZNF proteins, TALEN s and CRISPR /Cas9 or CRISPR /Cpf1, CRISPR / dC as9 or dC pf1, CRISPR 13a, topical delivery of mi RNA s and epigenetic memory have been successfully explored to MIR gene or mi RNA modulation and improve agronomic traits in several model or crop plants. However, advantages and drawbacks of each of these new biotechnological tools ( NBT s) are still not well understood. In this review, we provide a brief overview of the biogenesis and role of mi RNA s in response to abiotic or biotic stresses, we present critically the main NBT s used for the manipulation of MIR genes and mi RNA s, we show current efforts and findings with the MIR genes and mi RNA s modulation in plants, and we summarize the advantages and drawbacks of these NBT s and provide some alternatives to overcome. Finally, challenges and future perspectives to mi RNA modulating in important crops are also discussed.