Our group is involved in two different research lines: 1) Analysis of the plant response to spider mite feeding and, 2) Functional characterization of cysteine-proteases gene family and their inhibitors within an important crop such is barley.

1- The two-spotted spider mite Tetranychus urticae is a cosmopolitan agricultural acari pest feeding on over 1,100 different plants, 150 of them of agricultural importance, causing damages approaching 1 billion dollars worldwide (Figure 1). Due to high fecundity, inbreeding and short generation time Tetranychidae populations develop resistance to acaricides after one to four years of use. Our research pretends to establish the genetic basis of ability of mite to feed on a wide plant host, to understand the plant responses (genes and signalling pathways) that confer resistance to mite feeding and to apply the knowledge obtained through basic science as a potential new avenue for acari-pest control.

Figure 1. T. urticae on its silk and their effects on infected plant leaves.

2- Plant proteolysis is a complex metabolic process required for protein processing and turnover in which different protease classes play an essential role. We pretend to analyse the protease-inhibitor relationship within the cysteine-protease C1A and C13 families and their inhibitors (their cognate propeptides and cystatins) of barley and to elucidate their functions as partners in three different important physiological processes such as grain germination, leaf senescence and defence against pests.

Relevant results

Our team, as a member of the GAP-M consortium (Genomics in Agricultural Pest Management, has participated in the entire genome sequence and annotation of the spider mite T. urticae (Grbic et al. 2011). The consortium has performed a screen on arabidopsis, tomato and grape wine accessions to isolate the most resistant and sensitive ones. The selected accessions are being analyzed using the whole genome microarray to detect expression changes following spider mite feeding. In parallel, the mite transcriptome is being analyzed to determine how the host plants affect the pest. The databases and respective transcription profiles are being established for each combination of plant-spider mite. This resource could be used for the resistance breeding programs. Our consortium has also started the genome sequencing of other spider mite species extremely invasive, such id T. evansi which only feed Solanaceae species, and is a pest of recent introduction from South America in the EU agriculture. This new genome will also have a potential resource toward practical applications.

The most important results derived from our studies on barley cysteine-proteases and their modulators are related to the identification, molecular characterization and classification of barley cysteine-protease families C1A and C13, as targets of barley cystatins (Martinez et al., 2007; 2012). Bioinformatic approaches have allowed an evolutionary comparison of both protease classes and their inhibitors in representative species of different taxonomic groups (Martinez and Diaz, 2008; Cambra et al. 2010; Martinez, 2011). Gene expression patterns and sub-cellular localization have been determined and together with enzyme-cystatin interaction analyses, have demonstrated their specificity and the location where this interaction takes place in plant cell.

In addition, we have demonstrated that these peptidases have to be processed to become active, being their own propeptide important modulator of the peptidase activity (Cambra et al., 2012; submitted). Besides, the functional characterization of genes encoding cathepsin L- and F-like cysteine-proteases has shown their implication as partners in the mobilization of hordeins, the most important protein stored in the barley grain, as schemed in Figure 2 (Martinez et al., 2009), and probably in other physiological processes that are currently being studied. Experiments of over-expression and silencing proteases and/or inhibitor genes by generating transgenic barley plants are being carried out in collaboration with Dr. Kumlehn from the IPK-Gatersleben, Germany. The results will bring us new insights on their in vivo function.

Fig. 2. Scheme of the participation of cysteine-proteases and their modulators in the barley grain germination.

In addition, transgenic expression of selected cystatin genes in potato, maize and arabadiposis have been generated and in vitro and in vivo inhibitory tests have shown their potential as defence transgenes (Alvarez-Alfageme et al. 2007; Carrillo et al. 2011a, b y c). This work has been developed in collaboration with Felix Ortego´s group form the CIB-CSIC of Madrid, Spain.

Research Projects

-Pest gs anenomicd plant breeding in a sustainable agricutural pest management. ONTARIO MINISTRY OF RESEARCH AND INNOVATION, CANADA (AGL2) 2010-13.

-Caracterización molecular del hongo causante de la podredumbre carbonosa de la corona en fresa (Macrophomina phaseolina) en cultivos de Chile: desarrollo de estrategias biotecnológicas para su control. UPM (AL12-PID-04) 2012.

-Cisteín-proteasas de cebada: caracterización fisiológica de proteasas tipo papaína en funciones endógenas y defensa. MINECO (AGL11-23650) 2012-14.

Recent Publications

Martinez, M., Cambra, I., Santamaría, M.E., Diaz, I. 2012. “C1A Cysteine-proteases and their inhibitors in plants” Physiologia Plantarum doi: 10.1111/j.1399-3054.2012.01569.x

Martinez, M. 2011. “Plant protein-coding gene families: emerging bioinformatics approaches”. Trends in Plant Science 16 (10): 558-567

Carrillo, L., Herrero, I., Cambra, I., Sanchez-Monge R, Diaz, I., Martinez, M. 2011a. “Differential in vitro and in vivo effects of barley cysteine and serine protease inhibitors on phytpathohenic microorganisms”. Plant Physiology and Biochemistry 49: 1191-1200

Carrillo, L., Martinez, M., Alvarez-Alfageme, F., Castanera, P., Smagghe, G., Diaz, I., Ortego, F. 2011b. “A barley cysteine-protease inhibitor reduces the performance of two aphid species in artificial diets and transgenic arabidopsis pants”. Transgenic Research 20:305-319

Carrillo, L., Martinez, M., Ramessar, K., Cambra, I., Castanera, P., Ortego, F., Diaz, I. 2011c. “Expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteine-proteases”. Plant Cell Reports 30:101-112

Grbic, M., Van Leeuwen, T., Clark, R., Rombauts, S., Rouze, P., Grbić, V., Osborne, E., Dermauw, W., Thi Ngoc, P.C., Ortego, F., Hernandez-Crespo, P., Diaz, I., Martinez, M., et al. 2011. “The genome of Tetranychus urticae reveals herbivorous pest adaptations”. Nature 479:487-492

Martinez, M.; Cambra, I.; Carrillo, L.; Diaz-Mendoza, M.; Diaz, I. 2009."Characterization of the Entire cystatin gene family in barley and their target cathepsin L-like cysteine-proteases, partners in the hordein mobilization during seed germination". Plant Physiology 151(3):1531-1545

Martinez, M.; Diaz, I. 2008. "The origin and evolution of plant cystatins and their target cysteine proteinases indicate a complex functional relationship". BMC Evolutionary Biology. 8:198

Alvarez-Alfageme, F.; Martinez, M.; Pascual-Ruiz, S.; Castanera, P.; Diaz, I.; Ortego, F. 2007. "Effects of potato plants expressing a barley cystatin on the predatory bug Podisus maculiventris via herbivorous prey feeding on the plant". Transgenic research. 16(1):1-13

Martinez, M.; Diaz-Mendoza, M.; Carrillo, L.; Diaz, I. 2007. "Carboxy terminal extended phytocystatins are bifunctional inhibitors of papain and legumain cysteine proteinases". FEBS letters. 581(16):2914-2918