Exploring the potential of endophyte-plant interactions for improving crop sustainable yields in a changing climate.

Climate change poses a major threat to global food security, significantly reducing crop yields as cause of abiotic stresses, and for boosting the spread of new and old pathogens and pests. Sustainable crop management as a route to mitigation poses the challenge of recruiting an array of solutions and tools for the new aims. Among these, the deployment of positive interactions between the micro-biotic components of agroecosystems and plants can play a highly significant role, as part of the agro-ecological revolution. Endophytic microorganisms have emerged as a promising solution to tackle this challenge. Among these, Arbuscular Mycorrhizal Fungi (AMF) and endophytic bacteria and fungi have demonstrated their potential to alleviate abiotic stresses such as drought and heat stress, as well as the impacts of biotic stresses. They can enhance crop yields in a sustainable way also by other mechanisms, such as improving the nutrient uptake, or by direct effects on plant physiology. In this review we summarize and update on the main types of endophytes, we highlight several studies that demonstrate their efficacy in improving sustainable yields and explore possible avenues for implementing crop-microbiota interactions. The mechanisms underlying these interactions are highly complex and require a comprehensive understanding. For this reason, omic technologies such as genomics, transcriptomics, proteomics, and metabolomics have been employed to unravel, by a higher level of information, the complex network of interactions between plants and microorganisms. Therefore, we also discuss the various omic approaches and techniques that have been used so far to study plant-endophyte interactions.

Physiological Responses to Salt Stress at the Seedling Stage in Wild (Oryza rufipogon Griff.) and Cultivated (Oryza sativa L.) Rice.

Domesticated rice Oryza sativa L. is a major staple food worldwide, and the cereal most sensitive to salinity. It originated from the wild ancestor Oryza rufipogon Griff., which was reported to possess superior salinity tolerance. Here, we examined the morpho-physiological responses to salinity stress (80 mM NaCl for 7 days) in seedlings of an O. rufipogon accession and two Italian O. sativa genotypes, Baldo (mildly tolerant) and Vialone Nano (sensitive). Under salt treatment, O. rufipogon showed the highest percentage of plants with no to moderate stress symptoms, displaying an unchanged shoot/root biomass ratio, the highest Na+ accumulation in roots, the lowest root and leaf Na+/K+ ratio, and highest leaf relative water content, leading to a better preservation of the plant architecture, ion homeostasis, and water status. Moreover, O. rufipogon preserved the overall leaf carbon to nitrogen balance and photosynthetic apparatus integrity. Conversely, Vialone Nano showed the lowest percentage of plants surviving after treatment, and displayed a higher reduction in the growth of shoots rather than roots, with leaves compromised in water and ionic balance, negatively affecting the photosynthetic performance (lowest performance index by JIP-test) and apparatus integrity. Baldo showed intermediate salt tolerance. Being O. rufipogon interfertile with O. sativa, it resulted a good candidate for pre-breeding towards salt-tolerant lines.

The inability of barley to germinate after submergence depends on hypoxia-induced secondary dormancy.

Global climate change has dramatically increased flooding events, which have a strong impact on crop production. Barley (Hordeum vulgare) is one of the most important cereals and its cultivation includes a broad range of different environments. We tested the capacity to germinate of a large barley panel after a short period of submergence followed by a period of recovery. We demonstrate that sensitive barley varieties activate underwater secondary dormancy because of a lower permeability to oxygen dissolved in water. In sensitive barley accessions, secondary dormancy is removed by nitric oxide donors. The results of a genome-wide association study uncovered a Laccase gene located in a region of significant marker-trait association that is differently regulated during grain development and plays a key role in this process. Our findings will help breeders to improve the genetics of barley, thereby increasing the capacity of seeds to germinate after a short period of flooding.

Marker-Assisted Pyramiding of Blast-Resistance Genes in a japonica Elite Rice Cultivar through Forward and Background Selection.

Rice blast, caused by Pyricularia oryzae, is one of the main rice diseases worldwide. The pyramiding of blast-resistance (Pi) genes, coupled to Marker-Assisted BackCrossing (MABC), provides broad-spectrum and potentially durable resistance while limiting the donor genome in the background of an elite cultivar. In this work, MABC coupled to foreground and background selections based on KASP marker assays has been applied to introgress four Pi genes (Piz, Pib, Pita, and Pik) in a renowned japonica Italian rice variety, highly susceptible to blast. Molecular analyses on the backcross (BC) lines highlighted the presence of an additional blast-resistance gene, the Pita-linked Pita2/Ptr gene, therefore increasing the number of blast-resistance introgressed genes to five. The recurrent genome was recovered up to 95.65%. Several lines carrying four (including Pita2) Pi genes with high recovery percentage levels were also obtained. Phenotypic evaluations confirmed the effectiveness of the pyramided lines against multivirulent strains, which also had broad patterns of resistance in comparison to those expected based on the pyramided Pi genes. The developed blast-resistant japonica lines represent useful donors of multiple blast-resistance genes for future rice-breeding programs related to the japonica group.

Marker-Assisted Introgression of the Salinity Tolerance Locus Saltol in Temperate Japonica Rice.

BACKGROUND: Rice is one of the most salt sensitive crops at seedling, early vegetative and reproductive stages. Varieties with salinity tolerance at seedling stage promote an efficient growth at early stages in salt affected soils, leading to healthy vegetative growth that protects crop yield. Saltol major QTL confers capacity to young rice plants growing under salt condition by maintaining a low Na+/K+ molar ratio in the shoots. RESULTS: Marker-assisted backcross (MABC) procedure was adopted to transfer Saltol locus conferring salt tolerance at seedling stage from donor indica IR64-Saltol to two temperate japonica varieties, Vialone Nano and Onice. Forward and background selections were accomplished using polymorphic KASP markers and a final evaluation of genetic background recovery of the selected lines was conducted using 15,580 SNP markers obtained from Genotyping by Sequencing. Three MABC generations followed by two selfing, allowed the identification of introgression lines achieving a recovery of the recurrent parent (RP) genome up to 100% (based on KASP markers) or 98.97% (based on GBS). Lines with highest RP genome recovery (RPGR) were evaluated for agronomical-phenological traits in field under non-salinized conditions. VN1, VN4, O1 lines were selected considering the agronomic evaluations and the RPGR% results as the most interesting for commercial exploitation. A physiological characterization was conducted by evaluating salt tolerance under hydroponic conditions. The selected lines showed lower standard evaluation system (SES) scores: 62% of VN4, and 57% of O1 plants reaching SES 3 or SES 5 respectively, while only 40% of Vialone Nano and 25% of Onice plants recorded scores from 3 to 5, respectively. VN1, VN4 and O1 showed a reduced electrolyte leakage values, and limited negative effects on relative water content and shoot/root fresh weight ratio. CONCLUSION: The Saltol locus was successfully transferred to two elite varieties by MABC in a time frame of three years. The application of background selection until BC3F3 allowed the selection of lines with a RPGR up to 98.97%. Physiological evaluations for the selected lines indicate an improved salinity tolerance at seedling stage. The results supported the effectiveness of the Saltol locus in temperate japonica and of the MABC procedure for recovering of the RP favorable traits.

Effect of water management on microbial diversity and composition in an Italian rice field system.

Traditional rice cultivation consumes up to 2500 L of water per kg yield and new strategies such as the 'Alternate Wetting and Drying' (AWD) might be promising water-saving alternatives. However, they might have large impacts on the soil microbiology. In this study, we compared the bacterial and archaeal communities in experimental field plots, cultivated under continuously flooding (CF) and AWD management, by high-throughput sequencing of the 16S rRNA gene. We analysed alpha and beta diversity in bulk soil and on plant roots, in plots cultivated with two different rice cultivars. The strongest difference was found between soil and root communities. Beside others, the anaerobic methanotroph Methanoperedens was abundant in soil, however, we detected a considerable number of ANME-2a-2b on plant roots. Furthermore, root communities were significantly affected by the water management: Differential abundance analysis revealed the enrichment of aerobic and potentially plant-growth-promoting bacteria under AWD treatment, such as Sphingomonadaceae and Rhizobiaceae (both Alphaproteobacteria), and Bacteroidetes families. Microorganisms with an overall anaerobic lifestyle, such as various Delta- and Epsilonproteobacteria, and Firmicutes were depleted. Our study indicates that the bulk soil communities seem overall well adapted and more resistant to changes in the water treatment, whereas the root microbiota seems more vulnerable.

High accuracy of genome-enabled prediction of belowground and physiological traits in barley seedlings.

In plants, the study of belowground traits is gaining momentum due to their importance on yield formation and the uptake of water and nutrients. In several cereal crops, seminal root number and seminal root angle are proxy traits of the root system architecture at the mature stages, which in turn contributes to modulating the uptake of water and nutrients. Along with seminal root number and seminal root angle, experimental evidence indicates that the transpiration rate response to evaporative demand or vapor pressure deficit is a key physiological trait that might be targeted to cope with drought tolerance as the reduction of the water flux to leaves for limiting transpiration rate at high levels of vapor pressure deficit allows to better manage soil moisture. In the present study, we examined the phenotypic diversity of seminal root number, seminal root angle, and transpiration rate at the seedling stage in a panel of 8-way Multiparent Advanced Generation Inter-Crosses lines of winter barley and correlated these traits with grain yield measured in different site-by-season combinations. Second, phenotypic and genotypic data of the Multiparent Advanced Generation Inter-Crosses population were combined to fit and cross-validate different genomic prediction models for these belowground and physiological traits. Genomic prediction models for seminal root number were fitted using threshold and log-normal models, considering these data as ordinal discrete variable and as count data, respectively, while for seminal root angle and transpiration rate, genomic prediction was implemented using models based on extended genomic best linear unbiased predictors. The results presented in this study show that genome-enabled prediction models of seminal root number, seminal root angle, and transpiration rate data have high predictive ability and that the best models investigated in the present study include first-order additive × additive epistatic interaction effects. Our analyses indicate that beyond grain yield, genomic prediction models might be used to predict belowground and physiological traits and pave the way to practical applications for barley improvement.

The rice foot rot pathogen Dickeya zeae alters the in-field plant microbiome.

Studies on bacterial plant diseases have thus far been focused on the single bacterial species causing the disease, with very little attention given to the many other microorganisms present in the microbiome. This study intends to use pathobiome analysis of the rice foot rot disease, caused by Dickeya zeae, as a case study to investigate the effects of this bacterial pathogen to the total resident microbiome and to highlight possible interactions between the pathogen and the members of the community involved in the disease process. The microbiome of asymptomatic and the pathobiome of foot-rot symptomatic field-grown rice plants over two growing periods and belonging to two rice cultivars were determined via 16S rRNA gene amplicon sequencing. Results showed that the presence of D. zeae is associated with an alteration of the resident bacterial community in terms of species composition, abundance and richness, leading to the formation of microbial consortia linked to the disease state. Several bacterial species were significantly co-presented with the pathogen in the two growing periods suggesting that they could be involved in the disease process. Besides, culture-dependent isolation and in planta inoculation studies of a bacterial member of the pathobiome, identified as positive correlated with the pathogen in our in silico analysis, indicated that it benefits from the presence of D. zeae. A similar microbiome/pathobiome experiment was also performed in a symptomatically different rice disease evidencing that not all plant diseases have the same consequence/relationship with the plant microbiome. This study moves away from a pathogen-focused stance and goes towards a more ecological perception considering the effect of the entire microbial community which could be involved in the pathogenesis, persistence, transmission and evolution of plant pathogens.

Genomic Prediction of Grain Yield in a Barley MAGIC Population Modeling Genotype per Environment Interaction.

Multi-parent Advanced Generation Inter-crosses (MAGIC) lines have mosaic genomes that are generated shuffling the genetic material of the founder parents following pre-defined crossing schemes. In cereal crops, these experimental populations have been extensively used to investigate the genetic bases of several traits and dissect the genetic bases of epistasis. In plants, genomic prediction models are usually fitted using either diverse panels of mostly unrelated accessions or individuals of biparental families and several empirical analyses have been conducted to evaluate the predictive ability of models fitted to these populations using different traits. In this paper, we constructed, genotyped and evaluated a barley MAGIC population of 352 individuals developed with a diverse set of eight founder parents showing contrasting phenotypes for grain yield. We combined phenotypic and genotypic information of this MAGIC population to fit several genomic prediction models which were cross-validated to conduct empirical analyses aimed at examining the predictive ability of these models varying the sizes of training populations. Moreover, several methods to optimize the composition of the training population were also applied to this MAGIC population and cross-validated to estimate the resulting predictive ability. Finally, extensive phenotypic data generated in field trials organized across an ample range of water regimes and climatic conditions in the Mediterranean were used to fit and cross-validate multi-environment genomic prediction models including G×E interaction, using both genomic best linear unbiased prediction and reproducing kernel Hilbert space along with a non-linear Gaussian Kernel. Overall, our empirical analyses showed that genomic prediction models trained with a limited number of MAGIC lines can be used to predict grain yield with values of predictive ability that vary from 0.25 to 0.60 and that beyond QTL mapping and analysis of epistatic effects, MAGIC population might be used to successfully fit genomic prediction models. We concluded that for grain yield, the single-environment genomic prediction models examined in this study are equivalent in terms of predictive ability while, in general, multi-environment models that explicitly split marker effects in main and environmental-specific effects outperform simpler multi-environment models.

Analysis of Cadmium Root Retention for Two Contrasting Rice Accessions Suggests an Important Role for OsHMA2.

Two rice accessions, Capataz and Beirao, contrasting for cadmium (Cd) tolerance and root retention, were exposed to a broad range of Cd concentrations (0.01, 0.1, and 1 µM) and analyzed for their potential capacity to chelate, compartmentalize, and translocate Cd to gain information about the relative contribution of these processes in determining the different pathways of Cd distribution along the plants. In Capataz, Cd root retention increased with the external Cd concentration, while in Beirao it resulted independent of Cd availability and significantly higher than in Capataz at the lowest Cd concentrations analyzed. Analysis of thiol accumulation in the roots revealed that the different amounts of these compounds in Capataz and Beirao, as well as the expression levels of genes involved in phytochelatin biosynthesis and direct Cd sequestration into the vacuoles of the root cells, were not related to the capacity of the accessions to trap the metal into the roots. Interestingly, the relative transcript abundance of OsHMA2, a gene controlling root-to-shoot Cd/Zn translocation, was not influenced by Cd exposure in Capataz and progressively increased in Beirao with the external Cd concentration, suggesting that activity of the OsHMA2 transporter may differentially limit root-to-shoot Cd/Zn translocation in Capataz and Beirao.

Characterization of the Resistance to Powdery Mildew and Leaf Rust Carried by the Bread Wheat Cultivar Victo.

Leaf rust and powdery mildew are two important foliar diseases in wheat. A recombinant inbred line (RIL) population, obtained by crossing two bread wheat cultivars ('Victo' and 'Spada'), was evaluated for resistance to the two pathogens at seedling stage. Upon developing a genetic map of 8726 SNP loci, linkage analysis identified three resistance Quantitative Trait Loci (QTLs), with 'Victo' contributing the resistant alleles to all loci. One major QTL (QPm.gb-7A) was detected in response to Blumeria graminis on chromosome 7A, which explained 90% of phenotypic variation (PV). The co-positional relationship with known powdery mildew (Pm) resistance loci suggested that a new source of resistance was identified in T. aestivum. Two QTLs were detected in response to Puccinia triticina: a major gene on chromosome 5D (QLr.gb-5D), explaining a total PV of about 59%, and a minor QTL on chromosome 2B (QLr.gb-2B). A positional relationship was observed between the QLr.gb-5D with the known Lr1 gene, but polymorphisms were found between the cloned Lr1 and the corresponding 'Victo' allele, suggesting that QLr.gb-5D could represent a new functional Lr1 allele. Lastly, upon anchoring the QTL on the T. aestivum reference genome, candidate genes were hypothesized on the basis of gene annotation and in silico gene expression analysis.

Genome wide association studies for japonica rice resistance to blast in field and controlled conditions.

BACKGROUND: Rice blast, caused by the fungus Pyricularia oryzae, represents the most damaging fungal disease of rice worldwide. Utilization of rice resistant cultivars represents a practical way to control the disease. Most of the rice varieties cultivated in Europe and several other temperate regions are severely depleted of blast resistance genes, making the identification of resistant sources in genetic background adapted to temperate environments a priority. Given these assumptions, a Genome Wide Association Study (GWAS) for rice blast resistance was undertaken using a panel of 311 temperate/tropical japonica and indica accessions adapted to temperate conditions and genotyped with 37,423 SNP markers. The panel was evaluated for blast resistance in field, under the pressure of the natural blast population, and in growth chamber, using a mixture of three different fungal strains. RESULTS: The parallel screening identified 11 accessions showing high levels of resistance in the two conditions, representing potential donors of resistance sources harbored in rice genotypes adapted to temperate conditions. A general higher resistance level was observed in tropical japonica and indica with respect to temperate japonica varieties. The GWAS identified 14 Marker-Traits Associations (MTAs), 8 of which discovered under field conditions and 6 under growth chamber screening. Three MTAs were identified in both conditions; five MTAs were specifically detected under field conditions while three for the growth chamber inoculation. Comparative analysis of physical/genetic positions of the MTAs showed that most of them were positionally-related with cloned or mapped blast resistance genes or with candidate genes whose functions were compatible for conferring pathogen resistance. However, for three MTAs, indicated as BRF10, BRF11-2 and BRGC11-3, no obvious candidate genes or positional relationships with blast resistance QTLs were identified, raising the possibility that they represent new sources of blast resistance. CONCLUSIONS: We identified 14 MTAs for blast resistance using both field and growth chamber screenings. A total of 11 accessions showing high levels of resistance in both conditions were discovered. Combinations of loci conferring blast resistance were identified in rice accessions adapted to temperate conditions, thus allowing the genetic dissection of affordable resistances present in the panel. The obtained information will provide useful bases for both resistance breeding and further characterization of the highlighted resistance loci.

Correction to: Identification and mapping of expressed genes associated with the 2DL QTL for fusarium head blight resistance in the wheat line Wuhan 1.

Following publication of the original article [1], we have been notified that some important information was omitted by the authors in the Copyright note. The Copyright note should read as below.

GWAS for Starch-Related Parameters in Japonica Rice (Oryza sativa L.).

Rice quality is mainly related to the following two starch components, apparent amylose content (AAC) and resistant starch (RS). The former affects grain cooking properties, while RS acts as a prebiotic. In the present study, a Genome Wide Association Scan (GWAS) was performed using 115 rice japonica accessions, including tropical and temperate genotypes, with the purpose of expanding the knowledge of the genetic bases affecting RS and AAC. High phenotypic variation was recorded for the two traits, which positively correlated. Moreover, both the parameters correlated with seed length (positive correlation) and seed width (negative correlation). A correlational selection according to human preferences has been hypothesized for the two starch traits and grain size. In addition, human selection has been proposed as the causal agent even for the different phenotypes related to starch and grain size showed by the tropical and temperate japonica accessions utilized in this study. The present GWAS led to the identification of 11 associations for RS on seven chromosomes and five associations for AAC on chromosome 6. Candidate genes and co-positional relationships with quantitative trait loci (QTLs) previously identified as affecting RS and AAC were identified for 6 associations. The candidate genes and the new RS- and/or AAC-associated regions detected provide valuable sources for future functional characterizations and for breeding programs aimed at improving rice grain quality.

Effects of water management and cultivar on carbon dynamics, plant productivity and biomass allocation in European rice systems.

Water saving techniques, such as alternate wetting and drying (AWD), are becoming a necessity in modern rice farming because of climate change mitigation and growing water use scarcity. Reducing water can vastly reduce methane (CH4) emissions; however, this net climate benefit may be offset by enhanced carbon dioxide (CO2) emissions from soil. The main aims of this study were: to determine the effects of AWD on yield and ecosystem C dynamics, and to establish the underlying mechanistic basis for observed trends in net ecosystem C gain or loss in an Italian rice paddy. We investigated the effects of conventional water management (i.e. conventionally flooded paddy; CF) and AWD on biomass accumulation (aboveground, belowground, grain), key ecosystem C fluxes (net ecosystem exchange (NEE), net primary productivity (NPP), gross primary productivity (GPP), ecosystem respiration (ER), autotrophic respiration (RA), heterotrophic respiration (RH)), and soil organic matter (SOM) decay for four common commercial European rice cultivars. The most significant finding was that neither treatment nor cultivar affected NEE, GPP, ER or SOM decomposition. RA was the dominant contributor to ER for both CF and AWD treatments. Cultivar and treatment affected the total biomass of the rice plants; specifically, with greater root production in CF compared to AWD. Importantly, there was no effect of treatment on the overall yield for any cultivar. Possibly, the wetting-drying cycles may have been insufficient to allow substantial soil C metabolism or there was a lack of labile substrate in the soil. These results imply that AWD systems may not be at risk of enhancing soil C loss, making it a viable solution for climate change mitigation and water conservation. Although more studies are needed, the initial outlook for AWD in Europe is positive; with no net loss of soil C from SOM decomposition, whilst also maintaining yield.

Identification and mapping of expressed genes associated with the 2DL QTL for fusarium head blight resistance in the wheat line Wuhan 1.

BACKGROUND: Fusarium head blight (FHB) is a problem of great concern in small grain cereals, especially wheat. A quantitative trait locus (QTL) for FHB resistance (FHB_SFI) located on the long arm of chromosome 2D in the spring wheat genotype Wuhan 1 is a resistance locus which has potential to improve the FHB resistance of bread wheat since it confers effective resistance to wheat breeding lines. Recently, differentially expressed genes (DEG) have been identified by comparing near isogenic lines (NIL) carrying the susceptible and resistant alleles for the 2DL QTL, using RNA-Seq. In the present study, we aimed to identify candidate genes located within the genetic interval for the 2DL QTL for FHB resistance, as assessed by single floret inoculation (FHB_SFI), and possibly contributing to it. RESULTS: Combining previous and additional bioinformatics analyses, 26 DEG that were located on chromosome arm 2DL were selected for further characterization of their expression profile by RT-qPCR. Seven of those DEG showed a consistent differential expression profile between either three pairs of near isogenic lines or other genotypes carrying the R and S alleles for the 2DL QTL for FHB resistance. UN25696, which was identified in previous expression work using microarray was also confirmed to have a differential expression pattern. Those eight candidate genes were further characterized in 85 lines of a double haploid mapping population derived from the cross Wuhan 1/Nyubai, the population where the 2DL QTL was originally identified. The expression QTL for gene Traes_2DL_179570792 overlapped completely with the mapping interval for the 2DL QTL for FHB_SFI while the expression QTL for UN25696 mapped near the QTL, but did not overlap with it. None of the other genes had a significant eQTL on chromosome 2DL. Higher expression of Traes_2DL_179570792 and UN25696 was associated with the resistant allele at that locus. CONCLUSIONS: Of the 26 DEG from the 2DL chromosome further characterized in this study, only two had an expression QTL located in or near the interval for the 2DL QTL. Traes_2DL_179570792 is the first expression marker identified as associated with the 2DL QTL.

Dissection of coleoptile elongation in japonica rice under submergence through integrated genome-wide association mapping and transcriptional analyses.

Rice is unique among cereals for its ability to germinate not only when submerged but also under anoxic conditions. Rice germination under submergence or anoxia is characterized by a longer coleoptile and delay in radicle emergence. A panel of temperate and tropical japonica rice accessions showing a large variability in coleoptile length was used to investigate genetic factors involved in this developmental process. The ability of the Khao Hlan On rice landrace to vigorously germinate when submerged has been previously associated with the presence of the trehalose 6 phosphate phosphatase 7 (TPP7) gene. In this study, we found that, in the presence of TPP7, polymorphisms and transcriptional variations of the gene in coleoptile tissue were not related to differences in the final coleoptile length under submergence. In order to find new chromosomal regions associated with the different ability of rice to elongate the coleoptile under submergence, we used genome-wide association study analysis on a panel of 273 japonica rice accessions. We discovered 11 significant marker-trait associations and identified candidate genes potentially involved in coleoptile length. Candidate gene expression analyses indicated that japonica rice genotypes possess complex genetic elements that control final coleoptile length under low oxygen.

Regulation and Evolution of NLR Genes: A Close Interconnection for Plant Immunity.

NLR (NOD-like receptor) genes belong to one of the largest gene families in plants. Their role in plants' resistance to pathogens has been clearly described for many members of this gene family, and dysregulation or overexpression of some of these genes has been shown to induce an autoimmunity state that strongly affects plant growth and yield. For this reason, these genes have to be tightly regulated in their expression and activity, and several regulatory mechanisms are described here that tune their gene expression and protein levels. This gene family is subjected to rapid evolution, and to maintain diversity at NLRs, a plethora of genetic mechanisms have been identified as sources of variation. Interestingly, regulation of gene expression and evolution of this gene family are two strictly interconnected aspects. Indeed, some examples have been reported in which mechanisms of gene expression regulation have roles in promotion of the evolution of this gene family. Moreover, co-evolution of the NLR gene family and other gene families devoted to their control has been recently demonstrated, as in the case of miRNAs.