**3. Identification of genes involved with anthracnose resistance**

The pathogenic system *Colletotrichum lindemuthianum/Phaseolus vulgaris* has been studied as a model for almost one century [36] and, its infection mechanisms and disease development were extensively studied in the 1980s [37, 38, 39]. This species of *Colletotrichum* is one of the most studied due to its economic importance, infection strategy [38], ease of *in vitro* cultivation [40], and availability of an efficient and reproducible transformation system [41]. As a model system for plant/fungi interaction, it can provide valuable information in several aspects, like plant defense responses, phytoalexins, fungal-degrading cell wall enzymes, differentiation of fungal infection structures.

The susceptible common bean cultivars establish an interaction of compatibility with this fungus, what allows the development of the anthracnose disease, strongly affecting production and yield of beans; furthermore, this fungus has great variability and many races identified [42, 43]. With this, the genetic resistance is an important way of disease control. Genetic studies indicate that the common bean resistance to the anthracnose is related to multi-allelic loci [44, 45], which mostly comprise dominantly inherited genes denominated *Co* [45]. Bean cultivars resistant to anthracnose containing *Co* gene (s) respond to pathogen inoculation with an incompatible interaction. This interaction initiates with the pathogenic fungus inoculation, causing physiological variations and rapid changes in gene expression that activate defense responses in the host plant. Necrotrophic points, typical of a hypersensitive reaction (HR), occur at the infection site, resulting in a limited fungal growth. The HR, considered the primary response of the plant to the pathogen attack, is characterized by an oxidative burst due to the formation of reactive oxygen species (ROS) [46]. This initial plant response can be considered definitive in the determination of resistance to the pathogenic agent.

United States Department of Agriculture (USDA), found on http://iapreview.ars.usda.gov. Brazil has held a very significant collection of landraces and domesticated beans at EMBRAPA Arroz e Feijão and also at the Agronomic Institute of Campinas, which has been developing several new commercial varieties (http://www.iac.sp.gov.br/areasdepesquisa/graos/ feijao.php). Much more details about bean collections are found on Genesys (https:// www.genesys-pgr.org/welcome), a portal to information about Plant Genetic Resources for Food and Agriculture, describing many bean accessions and the places where they are kept. These collections comprise a very rich source of genetic materials that possess several features to be exploited in functional genomic and molecular breeding studies for the species. Among the genetic resources available are wild beans, landraces, breeding lines, recombinant inbred

populations, all distinguished between the Andean and Mesoamerican gene pools.

**3. Identification of genes involved with anthracnose resistance**

The pathogenic system *Colletotrichum lindemuthianum/Phaseolus vulgaris* has been studied as a model for almost one century [36] and, its infection mechanisms and disease development were extensively studied in the 1980s [37, 38, 39]. This species of *Colletotrichum* is one of the most studied due to its economic importance, infection strategy [38], ease of *in vitro* cultivation [40], and availability of an efficient and reproducible transformation system [41]. As a model system for plant/fungi interaction, it can provide valuable information in several aspects, like plant defense responses, phytoalexins, fungal-degrading cell wall enzymes, differentiation of

The susceptible common bean cultivars establish an interaction of compatibility with this fungus, what allows the development of the anthracnose disease, strongly affecting production and yield of beans; furthermore, this fungus has great variability and many races identified [42, 43]. With this, the genetic resistance is an important way of disease control. Genetic studies

A recent publication showed the work that has been done for many years to sequence the genome of the common bean, whose assembly has been made public by a consortium between the USDA-NIFA project "A sequence map of the common bean genome for bean improve‐ ment" and DOE-JGI and ARRA (Phaseolus vulgaris v1.0 – http://phytozome.jgi.doe.gov/). In total, 472.5 Mb of the 587-Mb genome were assembled and 98% of the sequence were geneti‐ cally anchored on the 11 chromosomes, using a SNP high-density map (7,015 markers) genotyped in the RIL (recombinant inbred lines) population derived from the cross Stampede × Red Hawk and another map with 261 SSRs and a set of Infinium markers. The 472.5 Mb were arranged in 41,391 contigs (~9.32% gap) and the annotation revealed 27,197 total proteincoding genes and 31,638 protein-coding transcripts, resulting in 4,441 total alternatively spliced transcripts [23]. The publication of this genome opened a series of new resources for developing research in many fields such as the mechanisms involved in biotic and abiotic

**2.3.** *Phaseolus vulgaris* **– The genome**

124 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

stresses in common bean.

fungal infection structures.

In the compatible interaction, the establishment of the pathogen in the plant tissue is aided by the production, by the fungus, of virulence effectors induced by the host [47, 48]. The life strategy adopted by the fungus (hemibiotrophic) make infected tissues remain without outward symptoms for up to three or more days [49, 50], and only after the entrance in the necrotrophic phase cause plant cell death and emergency of pathogenic lesions.

Despite the multi-allelic resistance already described for the common bean, new sources of resistance should always be searched due to the high variability among pathogen populations and occurrence of newly evolved virulent races. Furthermore, knowing the molecular pathways involved with the process of resistance in the plant can enable the transference of important genes to susceptible cultivars.

Common bean is not a species prone to be genetically transformed, although there is already a transgenic cultivar resistant to the Golden Mosaic Virus [51]. Furthermore, the genome of common bean was made available only recently, and reverse genetics through the use of mutant lines is still difficult due to few resources. Then, transcriptomic analysis appears as a suitable method to investigate the changes in gene expression in a plant under any kind of stress.

#### **3.1. Gene expression profiles from an incompatible interaction**

Studying gene expression profiles of incompatible interactions between *Phaseolus vulgaris* and *Colletotrichum lindemuthianum* may be an advantageous strategy to identify genes involved with anthracnose resistance because it can provide a direct answer about the potential modulations occurring in metabolic processes during an infection event with a resistance response by the host.

The first study devoted to generate a unigene data set of common bean using ESTs sequencing was described by [52], through the analysis of three EST libraries from the cultivar SEL 1308, consisting of 19-day-old trifoliate leaves, 10-day-old stem shoots, and 13-day-old stem shoots inoculated with the race 73 of *C. lindemuthianum* in an incompatible interaction. At that time, a total of 5,255 ESTs were sequenced, 2,332 from inoculated stem shoots, with 1,583 unigenes assigned for this library. More recently, [53] used this database to select candidate genes based on the number of ESTs found per unigene (or tentative contig) in each library, to study expression profiles in temporal and spatial scales during fungus infection. Twelve genes were chosen and tested in leaves, hypocotyls and epicotyls inoculated with *C. lindemuthianum* (Figure 1).

**Figure 1.** Relative expression (RE) ratio of 12 pathogenesis resistance-related transcripts in leaves (A), epicotyls (B), and hypocotyls (C) of common bean genotype SEL 1308 at 24, 48, 72, and 96 HAI with the race 73 of *C. lindemuthiaum*. Non-inoculated tissue was used as control for expression levels to determine the RE ratio. The symbol \* above the bars indicates statistical significance calculated using the Pair Wise Fixed Reallocation Randomization Test with *P* ≤ 0.05. (Extracted from [53]).

All genes showed modulation during this incompatible interaction. Some of them were rapidly activated and kept this activation, like PR1a, PR1b (known as good molecular markers for SAR (systemic acquired resistance)), and PR2 (a b-1,3-glucanase) (Figure 1), which act in plant defense by hydrolysing the cell walls of the fungal pathogens. All the others showed a variety of expression patterns according to time and tissue, for instance, PR16 proteins (germin-like), which were upregulated early in leaves and then fall down, and in epicotyls and hypocotyls only PR16b was upregulated in late periods of analysis (Figure 1). This kind of study not only give us an idea of the kinetics of induced defense responses of common bean against the anthracnose fungus but also can be used as a base line for others studies of resistance against a broad range of pathogens [53]. Furthermore, this work revealed differential and specific transcriptional profiles in different tissues of common bean, where specific defense processes may occur to contain the development of a pathogen. For more details, see [53].

chosen and tested in leaves, hypocotyls and epicotyls inoculated with *C. lindemuthianum*

126 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

**Figure 1.** Relative expression (RE) ratio of 12 pathogenesis resistance-related transcripts in leaves (A), epicotyls (B), and hypocotyls (C) of common bean genotype SEL 1308 at 24, 48, 72, and 96 HAI with the race 73 of *C. lindemuthiaum*. Non-inoculated tissue was used as control for expression levels to determine the RE ratio. The symbol \* above the bars indicates statistical significance calculated using the Pair Wise Fixed Reallocation Randomization Test with *P* ≤ 0.05.

All genes showed modulation during this incompatible interaction. Some of them were rapidly activated and kept this activation, like PR1a, PR1b (known as good molecular markers for SAR (systemic acquired resistance)), and PR2 (a b-1,3-glucanase) (Figure 1), which act in plant

(Figure 1).

(Extracted from [53]).

#### **3.2. The immune system model for** *Phaseolus vulgaris/ Colletotrichum lindemuthianum*

The innate immunity is a primitive way of defense against microbial infection shared by plants, insects, and animals. Differently from mammals that have mobile cells specialized in defense, each plant cell is responsible for its own defense. Thus, each cell integrates environmental signals in order to activate local and systemic defense responses.

The same EST libraries described before [52] were used by [54] to investigate global changes in gene expression of *P. vulgaris* inoculated with *C. lindemuthianum* in an incompatible interaction. In an extensive bioinformatics analysis, the ESTs were aligned by tBLASTX with the *Arabidopsis thaliana* (L.) Heynh genome, which is completely annotated and curated. With this, it was possible to conduct a functional comparison between the fungus-inoculated and the mock-inoculated library. Figure 2 shows the overall mechanisms found in this study. It was found that some processes involved with plant–pathogen interaction were upregulated in common bean in response to the presence of fungus, like defense response to fungus (GO: 0050832), regulation of defense response GO:0031347), regulation of response to stress (GO: 0080134), and stomatal movement (GO:0010118).

Response to cytokinin stimulus (GO:0009735) and ethylene-mediated signaling pathway (GO: 0009873) were upregulated, while jasmonic acid biosynthetic (GO:0031408) and metabolic (GO: 0009694) processes, as well as response to gibberellin stimulus (GO:0009739) and abscisic acidmediated signaling pathway (GO:0009738) were downregulated, indicating that there may be a hormonal control and cross-talk in common bean defense against *C. lindemuthianum*. According to [54], hormonal mechanisms can be used in some pathosystems for resistance and in others for susceptibility depending on the fungus life-style. While jasmonates (JA) were found to be important in disease susceptibility in Arabidopsis and tomato infected with *Pseudomonas syringae* [55, 56], a biotrophic bacterium, in common bean it is not used in signaling since *C. lindemuthianum* is a hemibiotrophic pathogen.

Still based on the analysis of ESTs libraries, infected common beans have its metabolism modulated for detoxification from ROS burst, once HR is occurring during the incompatible interaction; also, a downregulation of genes was observed related to plant development (organelle fission (GO:0048285), cell cycle process (GO:0022402), pattern specification process (GO:0007389), post-embryonic morphogenesis (GO:0009886), and regulation of post-embry‐ onic development (GO:0048580), typical of plants under stress that needs to reallocate resources to defense responses.

Finally, transcripts encoding for cell wall proteins showed an increase in abundance, suggest‐ ing that activities as cell wall modification, pathogen recognition, and transport and secretion of defense compounds are important in bean defense against anthracnose.

When looking for molecular components of the plant innate immunity (PTI – PAMP-triggered immunity or ETI – effector-triggered immunity), [54] observed that ETI (characterized by HR) can negatively regulate PTI. Transmembrane receptor protein tyrosine kinases and MAPKKK/ MEKK transcripts were significantly downregulated in fungus-inoculated library and this data validate by RT-qPCR.

**Figure 2.** A model of the bean innate immune system. The proposed model represents key molecular components and metabolic processes known to be involved in plant–pathogen interactions. Gene Ontology (GO) categories and En‐ zyme Codes (EC) inside boxes are differentially represented in the bean EST libraries (arrowheads pointing down rep‐ resent downregulation and arrowheads pointing up represent upregulation). Continuous arrows represent established relationship between components of the pathway and intermittent arrows represent undirected relationship. Compo‐ nents of PAMP-triggered immunity (PTI) are depicted to the right of the diagram. (Extracted from [54]).
