THE CRISPR/CAS9 EDITING OF A WRKY GENE AND THE OVEREXPRESSION OF A LIPOXYGENASE GENE FOR IMPROVING PATHOGEN RESISTANCE IN MAIZE

Fusarium verticillioides (Fv) is a major cereal pathogen causing stalk rot and ear rot in maize, negatively affecting crop productivity, and compromising food safety by producing the secondary metabolites fumonisins. Several studies were conducted to identify maize genes associated with host plant resistance to Fv infection and fumonisin accumulation. The maize WRKY transcription factors and the lipoxygenases (ZmLOXs) are well recognized as important players in plant defense against pathogens, and it is known that the host-pathogen lipid cross-talk influences the pathogenesis. In this regard, previous RNA-seq experiments reported the enhanced expression of ZmLOX genes in maize resistant genotypes and GWAS resulted in one SNP significantly associated with ZmWRKY125. The Clustered Regularly Interspaced Short Palindromic Repeat/associated Cas9 (CRISPR/Cas9) editing of ZmWRKY125 and the transgenic overexpression of ZmLOX4 genes were carried out to investigate the possible implication of these two genes in the resistance mechanisms against Fv. Before cloning experiments, protein domain conservation and different splicing products have been analyzed comparing homologues and orthologues for both genes. As regards ZmWRKY125, the CRISPR cloning was based on a double cloning using two different guides (sgRNA) for one gene target. Agrobacterium tumefaciens mediated transformation was used for introducing the construct under the maize promoter ZmpUBI in the binary vector p1609 in maize A188 line. Mutants from three different transformation events were obtained. For each event, T2 plants will be genotyped to find homozygous for the mutation that in turn will be phenotyped for Fv resistance and fumonisin content. As regards ZmLOX4, the gene was cloned under an overexpressed promoter involved in kernel development in the vector L1781, and the same transformation conditions adopted for the CRISPR/Cas9 editing of ZmWRKY125 were used. Mutants from two different transformation events were obtained. For each event, T2 plants were genotyped in order to find homozygous for the mutation. Homozygous plants will be further evaluated for Fv resistance, fumonisin accumulation, oxylipin content as well as for the expression analysis of the main genes involved in the jasmonic acid pathway

Assessing the genetic and molecular basis of resistance to Fusarium verticillioides in maize

Maize (Zea mays L.) is a major cereal crop, the second most cultivated crop in the world. Maize is used for human consumption, livestock feed, and biofuel. In addition to its economic importance, maize has been a widely used model species for genetics and plant biology. Among the limitations to maize production and seed quality, the several diseases caused by Fusarium are severe and largely diffused. Maize research has been oriented towards distinguishing the levels of resistance to ear rot caused by Fusarium verticillioides, however, it has not yet been possible to clarify the model of genetic action of the resistance that could guide the selection of resistant genotypes. The Multi-parent Advance Generation Intercross (MAGIC) maize population was previously used to identify quantitative trait loci (QTL) for Fusarium seedling rot resistance using the rolled towel assay (RTA) that allows fast and reliable phenotyping at early developmental stages. Production of transcriptomic data specific to the infection phase may increase the precision by which candidate genes are identified. RNA-Seq approach was used to compare the genome-wide gene expression patterns in maize scutella and early germinating shoots in the eight MAGIC maize founder lines in mock and F. verticilloides treated seeds. The RTAs were performed at 48, 72, 96, 120, 168 hours post inoculation (hpi) under two conditions control and treated to identify the appropriate time point for the investigation of MAGIC maize founder lines transcriptome profiles. Twenty seeds were used for each RTA in both treatments, in the treated, the seeds were inoculated with 100 \u3bcl of a 3.5 x 106 ml-1 spore suspension of F. verticillioides ITEM10027 (MPVP 294). Real-time PCR was applied on plant and pathogen specific genes to identify the best time point for RNA extraction, which turned out to be 72 hpi. RNA was extracted from the scutella and early germinating shoots and a total of 48 cDNA libraries (8 genotypes x 2 conditions x 3 biological replicates) have been produced and subjected to sequencing. Transcriptomic data on the parental lines will be projected onto recombinant inbred lines reconstructed genomes and used to narrow down QTL intervals to their genetic determinants. The defense-related transcriptional changes will shed light on and related them to the specific genomic regions identified by QTL mapping

DIFFERENTIAL MODULATION OF PLANT AND FUNGAL PHOSPHATE TRANSPORTERS AND EXPRESSION OF MYCELIAL TRAITS IN DIVERSE MYCORRHIZAL MAIZE INBRED LINES

Current food crop production systems are heavily dependent on the use of chemical fertilizers, of which those containing phosphorus (P), essential for plant growth and development, represent a non-renewable mineral resource whose reserves are estimated to decline within 100-200 years. The microbiota associated with plant roots, mainly represented by arbuscular mycorrhizal fungi (AMF) can be exploited for reducing external P input into agro-ecosystems. In this study, plant and fungal variables involved in P acquisition were investigated in four maize inbred lines (Oh40B, Mo17, Oh43 and B73), differing for mycorrhizal responsiveness and low-P tolerance, when inoculated with the symbiont Rhizoglomus irregulare. The expression patterns of genes encoding phosphate transporters (PTs) in extraradical and intraradical mycelium (ERM and IRM) and in maize roots were assessed along with plant growth responses, P uptake and ERM extent and structure. The four maize lines differed in expression levels of PT genes in both plant and fungal tissues, in ERM phenotypic traits and plant performance. Expression of PT genes in roots and ERM of the low-P tolerant maize line Mo17 was higher than that detected in the low-P susceptible line B73, which revealed larger ERM hyphal densities and interconnectedness. Significant correlations were found between ERM structural traits, and both expression levels of PT genes and mycorrhizal host benefit data. Further studies aimed at improving our knowledge of the genetic mechanisms regulating the functioning of AMF symbiosis may lead to the development of new strategies of targeted molecular breeding to obtain more sustainable low-P tolerant crops

Cell death induced by mycotoxin fumonisin B1 is accompanied by oxidative stress and transcriptional modulation in Arabidopsis cell culture

Key message: Fumonisin B1 induces rapid programmed cell death in Arabidopsis cells, oxidative and nitrosative bursts, and differentially modulates cell death responsive genes. Glutathione is the main antioxidant involved in the stress response. Abstract: Fumonisin B1 (FB1) is a fungal toxin produced by Fusarium spp. able to exert pleiotropic toxicity in plants. FB1 is known to be a strong inducer of the programmed cell death (PCD); however, the exact mechanism underling the plant–toxin interactions and the molecular events that lead to PCD are still unclear. Therefore, in this work, we provided a comprehensive investigation of the response of the model organism Arabidopsis thaliana at the nuclear, transcriptional, and biochemical level after the treatment with FB1 at two different concentrations, namely 1 and 5 µM during a time-course of 96 h. FB1 induced oxidative and nitrosative bursts and a rapid cell death in Arabidopsis cell cultures, which resembled a HR-like PCD event. Different genes involved in the regulation of PCD, antioxidant metabolism, photosynthesis, pathogenesis, and sugar transport were upregulated, especially during the late treatment time and with higher FB1 concentration. Among the antioxidant enzymes and compounds studied, only glutathione appeared to be highly induced in both treatments, suggesting that it might be an important stress molecule induced during FB1 exposure. Collectively, these findings highlight the complexity of the signaling network of A. thaliana and provide information for the understanding of the physiological, molecular, and biochemical responses to counteract FB1-induced toxicity

Characterization and Valorization of Maize Landraces from Aosta Valley

While there is a rich collection of maize germplasm from Italy, it lacks genetic resources from the Aosta Valley, an isolated mountain region where landraces have been preserved in the absence of modern germplasm introductions. These local materials, which are still cultivated mainly at household level, can have high importance from a genetic and historical point of view. In the present study, five landraces named, after the collecting sites, Arnad, Arnad-Crest, Chatillon, Entrebin and Perloz, were sampled in Aosta Valley and subjected to historic, morphologic and genetic characterization. This study provided evidence for the landraces' long presence in Aosta Valley, a significant genetic variability and differentiation among the investigated landraces. Globally, 67 different alleles were detected ranging from 4 for markers phi127 and p-bnlg176 to 10 for phi031, with a mean of 6.7 alleles per locus. Observed heterozygosity levels were comprised from 0.16 to 0.51 and are generalkly lower than expected heterozigosity supporting fixation at some loci. STRUCTURE analysis revealed clear separation between accessions revealing the presence of four ancestral populations. This may be explained by the long reproductive isolation experienced by these materials. Finally, morphological observations confirm the high diversity between landraces revealing that they generally have flint kernels, variable color from yellow to dark red (Chatillon) while Perloz showed kernels with an apical beak. The present work confirms the importance of mountain areas in conserving biodiversity and increases the rich Italian maize germplasm with materials well adapted to marginal areas. Such new genetic variability may be used to breed new materials for more resilient agriculture

FUNCTIONAL STUDY OF LIPOXYGENASE-MEDIATED RESISTANCE AGAINST ASPERGILLUS FLAVUS AND FUSARIUM VERTICILLIOIDES INFECTION IN MAIZE

Mycotoxin contamination of maize kernels by fungal pathogens Aspergillus flavus (Af) and Fusarium verticillioides (Fv) is a chronic global challenge impacting food security, health and trade. Current disease management practices are proven inadequate and strategies gearing towards hostmediated resistance can be an effective and sustainable approach to strengthen efforts to control the pathogens. Lipid modification into a diverse array of compounds, oxylipins, are recognized in response of plants to fungal pathogens. Lipoxygenase genes (LOXs) play a crucial role in the enzymatic oxidation of polyunsaturated fatty acids (PUFAs) into 9(S)- and 13(S)-hydroperoxides that are further metabolized into different oxylipins including jasmonates, oxo- and keto-fatty acids and volatiles. The involvement of maize LOXs (ZmLOXs) in this respect has been a subject of studies and their genetic manipulation resulted in the alteration of resistance or susceptibility to fungal pathogens in maize. However, the maize genome encodes six 9-LOX and seven 13-LOX isoforms and the specific role of each isoform remains elusive. The current study investigated the role of ZmLOXs in host resistance against the fungi Af and Fv using in silico and in planta approaches. The phylogenetic relationship, sequence similarity, protein domain structures, and transcript level structural variations were explored by comparing publicly available maize pan-genomes. Furthermore, the role of ZmLOXs against Af and Fv infection was investigated through their expression analysis along with further key genes involved in oxylipin biosynthesis, mycotoxin accumulation and lipid profiles in a ZmLOX4 mutant line (UFMulox4) together with W22, Mo17 and Tzi18 inbred lines at 3- and 7-days post-inoculation (dpi). ZmLOX proteins showed considerable variations in their sequences, functional domain structure and transcript structural variations among the pan-genome members. Among the studied lines, Tzi18 showed the highest resistance to the pathogens coupled with the lowest mycotoxin accumulation, while the mutant, UFMulox4, was highly susceptible to both pathogens with the most elevated content of mycotoxins. Fv inoculation determined a stronger induction of ZmLOXs and maize allene oxide synthase genes as compared to Af . Higher constitutive levels of 9-LOXs genes, ZmLOX1 and ZmLOX2 and an induction of ZmLOX4 were recorded in Tzi18, while the upregulation of ZmLOX1 and ZmLOX4 observed in Mo17 might have larger active role in resistance against Fv. Liquid chromatography-mass spectrometry further revealed an increased accumulation of the linoleic (18:2) derived 9-cyclopentenone, 10- oxo-11-phytoenoic acid (10-OPEA), in Fv inoculated kernels of Tzi18 and Mo17, which was previously identified to inhibit fungal growth in vitro. Hence, the results confirm the pivotal role of ZmLOXs in controlling the resistance mechanisms against these two pathogens

TRANSCRIPTIONAL ANALYSIS OF EIGHT MAGIC MAIZE PARENTAL LINES INFECTED WITH FUSARIUM VERTICILLIOIDES

Maize (Zea mays L.) is among the most important crops worldwide for food, feed, biofuels, and industrial applications. Its cultivation faces significant constraints due to Fusarium species that affect the quality and quantity of maize products. Among these, Fusarium verticillioides is responsible for severe diseases including seedling blights, stalk rot, and ear rot. The impact of the fungus is worsened by the fact that chemical and agronomic measures used to control Fusarium infection are often inefficient. Hence, genetic resistance is considered the most reliable resource to reduce damages caused by F. verticillioides. This study aims to elucidate the genetic basis of resistance to this fungus in maize. Young seedlings of eight divergent maize lines, founder of the MAGIC population, were artificially inoculated with a F. verticillioides strain using the rolled towel assay method. Total RNA was extracted from both control and treated samples after 72 hours of artificial inoculation and underwent paired-end sequenced with Illumina technology. Here we report the use this large transcriptomic dataset to identify the early transcriptional changes and the differentially expressed genes (DEGs) involved in fungal infection. The analysis identified several hundred DEGs, whose functions were explored through Gene Ontology enrichment analysis. A co-expression network analysis further refined the set of genes with potential implications in disease response. The results identify a limited set of genes that might play an important roles in maize resistance to F. verticillioides providing new insights into the molecular resistance mechanisms against the pathogen

CHARACTERIZATION AND VALORIZATION OF MAIZE LANDRACES FROM VALLE D'AOSTA

During 1949-1950 in Italy begun a formal investigation to characterize maize (Zea mays L.) cultivation. In 1954, started a project for the sampling of all Italian maize landraces; this work ended with the collection of 562 different accessions collected in all regions with the exception of Valle d’Aosta, even if historical cultivation of maize in this Region is well documented. In Italy maize landraces have been extensively grown until the mid of the XX century when the cultivation of hybrid took place due to their significant agronomic performances. Despite that, being Valle d’Aosta a mountain region where intensive maize cultivation never started, it was possible to preserve the presence of some landraces. These local materials, which are still cultivated, mainly at domestic level, have high importance from a genetic and historical point of view. Recently, 5 maize landraces from Valle d’Aosta and 2 landraces from the adjacent Canavese (Piedmont) have been collected and subjected to historic, morphologic and genetic characterization. These landraces were named after the sampling location as it follows: Arnad, Arnad-Crest, Chatillon, Entrebin, Perloz, Bianco Canavese, and Rostrato Canavese. Firstly, on these 6 varieties the historic characterization has been carried out. Information and photographs have been searched in local archives and this was crucial to prove their long presence in all the sampling sites under study. From this historic reconstruction, the variety Entrebin resulted as the one that is better historically characterized. To study the variability and differentiation of landraces from Valle d’Aosta, the genetic characterization was performed by the means of 10 SSR markers tested on 20 samples from each landrace. This study highlighted a significant genetic variability among the landraces and, especially, a good level of differentiation between the accessions under investigation. This last result may be explained by the long reproductive isolation experienced by these materials. Complete morphological characterization is actually ongoing. Preliminary morphological observations revealed that these landraces have, generally, flint kernels with the exception of Bianco Canavese (dent) whose color is variable from white (Bianco Canavese) to dark red (Chatillon). Arnad landrace showed 8 kernel rows, probably being an Eight-rowed Flint while the others presented more rows, like many Derived Races. Interestingly, Perloz and Rostrato Canavese showed kernels with an apical beak which was more pronounced in the latter. This suggest that these two landraces belong to the “Rostrata” group, which is common in mountain areas. The present work confirms the importance of mountain areas in conserving biodiversity and increases the rich Italian maize germplasm with materials well adapted to marginal areas. Such new genetic variability may be used to breed new materials for a more resilient agriculture