Strategy

For many crop plants such as corn, beans, tomato (^{Hirsch and Buell, 2013}) among others, the determination of a whole genome sequence in order to carry out genetic analysis has been feasible, however, A. tequilana has a genome content of almost twice the size (4000Mb) (^{Palomino et al., 2007}) of corn and development and construction of a whole genome sequence is still technically challenging. Therefore, our initial strategy was to develop methods for transcriptome (RNAseq) analysis in agave species. Transcriptome analysis offers the advantages that less data needs to be generated and processed lowering costs. Additionally, data can be related to tissue types or developmental stages providing information on expression levels of specific genes under specific conditions. We have generated transcriptome data for A. tequilana, A. deserti, A. striata and A. victoria-reginae (^{Ávila de Dios et al., 2015}) and using this information have identified, cloned and characterized the cDNAs (coding sequences) responsible for the regulation of fructan metabolism and some members of a family of genes involved in the regulation of flowering time in A. tequilana.

A total of 15 genes involved in fructan/sucrose metabolism were identified and classified as encoding fructan synthesizing enzymes, fructan degrading enzymes or invertases. Genetic transformation of agave species is still laborious and inefficient and therefore we are using heterologous systems such as P. pastoris and A. thaliana in order to functionally characterize the identified genes/enzymes. In silico expression patterns based on transcriptome data indicate which genes are expressed in specific tissues or during specific developmental stages indicating their relative importance in different processes or metabolism. Based on these data we have determined and compared expression patterns for each of the fructan metabolism genes and most of these in silico expression patterns have now been confirmed by qRT-PCR (^{Ávila de Dios et al., 2019}, ^{Pérez-López et al., 2021}).

By employing a similar strategy, genes from several families known to be involved in the regulation of flowering time in the model system A. thaliana (FT, gibberellin metabolism, MYB factors) were also identified in A. tequilana and based on in silico expression patterns a working model for regulation of flowering in A. tequilana was proposed (^{Ávila deDios et al., 2019}). Currently we are functionally characterizing in detail the A. tequilana FT family.

An example of preliminary omics analysis in Colletotrichum lindemuthianum

The C. lindemuthianum - common bean pathosystem involves a gene-for-gene interaction between resistance (R) genes in the common bean host cultivars and avirulence (avr) genes in the fungal pathogen. Previous work in our group identified many distinct C. lindemuthianum races based on the differential series of common bean cultivars (^{González et al., 1998}, ^{Mendoza et al., 2001}, ^{Rodríguez-Guerra et al., 2003}, ^{González-Chavira et al., 2004}). We initiated a transcriptome strategy to compare the differential gene expression patterns at the early stages of infection on the common bean cultivar BAT93 inoculated with either a virulent or a non-virulent race (Race 1088 and Race 256 respectively) of C. lindemuthianum (Figure 1 and Table 1). Details of the experimental strategy are shown in Table 2. RNAseq was carried out on samples from each time point (samples from 4-96 hours were pooled) (Table 3) and the data was analyzed and compared in order to eventually identify genes expressed differentially during compatible and incompatible interactions (Medina-Chavez and Simpson unpublished).

Figure 1 Microscopic analysis 7 days after inoculation of common bean seedlings with Colletotrichum lindemuthianum. In the upper left quadrant, the formation of reproductive structures (acervuli) by the pathogen is observed in the compatible interactions for both cultivars with strain 1088. On the right side for the same strain a large number of conidia and appresoria are shown. In the interactions with strain 256, a large number of hyphae are observed for the cultivar Victoria as well as the presence of young conidia on the necrotic tissue of the plant, whereas for the cultivar BAT 93, hypersensitive reactions caused by the defense response are observed with the localized death of cells in contact with the pathogen. 

Genomic and transcriptomic analyses are currently essential and cost-effective tools for genetic analysis and can easily be applied to plants and/or their pathogens. The P. vulgaris-C. lindemuthianum pathosystem is particularly amenable given the well -documented series of differential cultivars and corresponding C. lindemuthianum races. The recent release and ongoing analysis of several P. vulgaris genomes (^{Rendón-Anaya et al., 2017}) will greatly facilitate this research.