CRISPR technology towards genome editing of the perennial and semi-perennial crops citrus, coffee and sugarcane.

Gene editing technologies have opened up the possibility of manipulating the genome of any organism in a predicted way. CRISPR technology is the most used genome editing tool and, in agriculture, it has allowed the expansion of possibilities in plant biotechnology, such as gene knockout or knock-in, transcriptional regulation, epigenetic modification, base editing, RNA editing, prime editing, and nucleic acid probing or detection. This technology mostly depends on in vitro tissue culture and genetic transformation/transfection protocols, which sometimes become the major challenges for its application in different crops. Agrobacterium-mediated transformation, biolistics, plasmid or RNP (ribonucleoprotein) transfection of protoplasts are some of the commonly used CRISPR delivery methods, but they depend on the genotype and target gene for efficient editing. The choice of the CRISPR system (Cas9, Cas12), CRISPR mechanism (plasmid or RNP) and transfection technique (Agrobacterium spp., PEG solution, lipofection) directly impacts the transformation efficiency and/or editing rate. Besides, CRISPR/Cas technology has made countries rethink regulatory frameworks concerning genetically modified organisms and flexibilize regulatory obstacles for edited plants. Here we present an overview of the state-of-the-art of CRISPR technology applied to three important crops worldwide (citrus, coffee and sugarcane), considering the biological, methodological, and regulatory aspects of its application. In addition, we provide perspectives on recently developed CRISPR tools and promising applications for each of these crops, thus highlighting the usefulness of gene editing to develop novel cultivars.

Transcriptional patterns of Coffea arabica L. nitrate reductase, glutamine and asparagine synthetase genes are modulated under nitrogen suppression and coffee leaf rust.

This study evaluated the transcriptional profile of genes related to nitrogen (N) assimilation in coffee plants susceptible and resistant to rust fungi under N sufficiency and N suppression. For this purpose, we inoculated young coffee leaves with Hemileia vastatrix uredospores and collected them at 0, 12, 24 and 48 hours post-inoculation (HPI) to evaluate the relative expressions of genes encoding cytosolic glutamine synthetase (CaGS1 ), plastid glutamine synthetase (CaGS2 ), nitrate reductase (CaNR), and asparagine synthetase (CaAS). The genes exhibited distinct patterns of transcriptional modulation for the different genotypes and N nutritional regimes. The resistant genotype (I59) presented high levels of transcription in response to pathogen inoculation for CaNR and CaGS1 genes, evaluated under N sufficiency in the initial moments of infection (12 HPI). The gene CaGS1 also showed a peak at 48 HPI. The susceptible genotype (CV99) showed increased transcript rates of CaNR at 12 and 24 HPI in response to rust inoculation. The transcriptional patterns observed for CV99, under N suppression, were high levels for CaAS and CaGS2 at all post-inoculation times in response to coffee leaf rust disease. In addition, CaGS1 was up-regulated at 48 HPI for CV99. Cultivar I59 showed high transcript levels at 12 HPI for CaAS and peaks at 24 and 48 HPI for CaGS2 in inoculated samples. Consequently, total chlorophyl concentration was influenced by N suppression and by rust infection. Regarding enzyme activities in vitro for glutamine synthetase and CaNR, there was an increase in infected coffee leaves (I59) and under N sufficiency. Moreover, CV99 was modulated in both N nutritional regimes for GS activity in response to rust. Our results indicate that N transport genes trigger a differential modulation between genotypes through the action of rust disease.

Genome-wide association study reveals candidate genes influencing lipids and diterpenes contents in Coffea arabica L.

Lipids, including the diterpenes cafestol and kahweol, are key compounds that contribute to the quality of coffee beverages. We determined total lipid content and cafestol and kahweol concentrations in green beans and genotyped 107 Coffea arabica accessions, including wild genotypes from the historical FAO collection from Ethiopia. A genome-wide association study was performed to identify genomic regions associated with lipid, cafestol and kahweol contents and cafestol/kahweol ratio. Using the diploid Coffea canephora genome as a reference, we identified 6,696 SNPs. Population structure analyses suggested the presence of two to three groups (K = 2 and K = 3) corresponding to the east and west sides of the Great Rift Valley and an additional group formed by wild accessions collected in western forests. We identified 5 SNPs associated with lipid content, 4 with cafestol, 3 with kahweol and 9 with cafestol/kahweol ratio. Most of these SNPs are located inside or near candidate genes related to metabolic pathways of these chemical compounds in coffee beans. In addition, three trait-associated SNPs showed evidence of directional selection among cultivated and wild coffee accessions. Our results also confirm a great allelic richness in wild accessions from Ethiopia, especially in accessions originating from forests in the west side of the Great Rift Valley.

Large-scale analysis of differential gene expression in coffee genotypes resistant and susceptible to leaf miner-toward the identification of candidate genes for marker assisted-selection.

BACKGROUND: A successful development of herbivorous insects into plant tissues depends on coordination of metabolic processes. Plants have evolved complex mechanisms to recognize such attacks, and to trigger a defense response. To understand the transcriptional basis of this response, we compare gene expression profiles of two coffee genotypes, susceptible and resistant to leaf miner (Leucoptera coffella). A total of 22000 EST sequences from the Coffee Genome Database were selected for a microarray analysis. Fluorescence probes were synthesized using mRNA from the infested and non-infested coffee plants. Array hybridization, scanning and data normalization were performed using Nimble Scan® e ArrayStar® platforms. Genes with foldchange values +/-2 were considered differentially expressed. A validation of 18 differentially expressed genes was performed in infected plants using qRT-PCR approach. RESULTS: The microarray analysis indicated that resistant plants differ in gene expression profile. We identified relevant transcriptional changes in defense strategies before insect attack. Expression changes (>2.00-fold) were found in resistant plants for 2137 genes (1266 up-regulated and 873 down-regulated). Up-regulated genes include those responsible for defense mechanisms, hypersensitive response and genes involved with cellular function and maintenance. Also, our analyses indicated that differential expression profiles between resistant and susceptible genotypes are observed in the absence of leaf-miner, indicating that defense is already build up in resistant plants, as a priming mechanism. Validation of selected genes pointed to four selected genes as suitable candidates for markers in assisted-selection of novel cultivars. CONCLUSIONS: Our results show evidences that coffee defense responses against leaf-miner attack are balanced with other cellular functions. Also analyses suggest a major metabolic reconfiguration that highlights the complexity of this response.