Role of a secondary metabolism gene cluster in the pathogenic interaction between Aspergillus flavus and Zea mays

The genus Aspergillus includes different species with a wide diversity of lifestyle and different pathogenicity mode. Aspergillus is one of the best described group mainly because its economic importance and because these fungi are recognized as secondary metabolic products causing animal and human diseases (S. Aaike and N.P. Keller, 2011). Aspergillus flavus is studied for its role in producing aflatoxins, a secondary metabolites product known to be the most carcinogenic natural substances currently detected in food: that is why they represent a health (causing aspergillosis) and economic issue. A.flavus has been studied for years in comparison with A.fumigatus but early studies of invasive aspergillosis in non-immunocompromised murine models demonstrated how A.flavus is more virulent than almost other Aspergillus species; furthermore, A.flavus causes a broad range spectrum of disease in humans from cutaneous infection to central nervous system infection (Hedayati et al., 2007). Zea mays is one of the big-three crops in the world and it represents one of the best-characterized A. flavus host. Because of its importance, researches are concerning the problem of disease since plant pathogens, like Aspergillus flavus, and the presence of mycotoxins on crops. Multiple mechanism are likely involved in resistance to aflatoxin contamination (Dolezal et al., 2014). Aflatoxin production in corn is extremely variable due, in part, to its sporadic occurrence among ears and among kernels within an ear; fungal structures associated with aflatoxins such as conidial heads and sclerotia likely exhibit a similar sporadic distribution pattern between corn ear and kernel (Horn et al., 2013). For all those reasons, it’s become very important define the “mycotoxins problem” and control the aflatoxins levels in crop pre-harvest and post-harvest; to do that, it’s necessary a global comprehension of cell cycle and infection pathway of A.flavus to reduce the damages. Starting from previous studies aimed to identify gene clusters encoding for secondary metabolites, involved in pathogenicity of A. flavus, we focused on Cluster32 and specifically on a Zn2Cys6 transcription factors, present inside the cluster. Our purpose is to understand its role in the regulation of Cluster32 expression and to clarify finally its significance within the process of pathogenesis. To achieve this, we designed a knockout mutant for Zn2Cys6 via the TOPO cloning method: we have assembled a construct containing the argD gene, coding for the enzyme ACETYL ORNITHINE AMINOTRANSFERASE, flanked by 3'UTR and 5'UTR, regions homologue to Zn2Cys6. Once obtained, we used the deletion construct to transform AFC-1, a double auxotroph mutant incapable of producing Arginine and Uracil. Simultaneously, to characterize better the metabolic profile related to the cluster 32, we produced overexpression mutants of Zn2Cys6 fused to GFP. Thus, mutants were screened by fluorescence emission. Such mutant, have been tested to assay pathogenicity and fitness in different environmental conditions, compared to the wild type. In mining the genome of A. flavus for identifying secondary metabolite clusters putatively involved in the pathogenesis process, our attention has turned also to some fungal effectors such as salicylate hydroxylase, quercetinases and necrosis/ethylene inducing proteins (NepA) belonging to cluster 32. In order to understand how this cluster works during the disease, we conducted histological and histochemical experiments in A. flavus pin bar infected maize caryopses. Within the same samples, we check the expression of specific genes inside the cluster (e.g. salOH, nepA), monitor, by LC-MS/MS, the production of salicylate, and check for the presence of its dehydroxylated form, i.e. cathecol. Within this frame several mutants of A. flavus impaired or enhanced in specific functions (e.g. cluster32 overexpression, nepA KO and OE strains) were checked for their ability to cause disease in maize caryopses. It emerges a scenario in which fungal progression through living tissues (e.g. aleuron) is accompanied by a significant arise in the levels of fungal effectors such as SalOH and nepA, the degradation of SA that, in turn, appear strategical for the fungus to bypass caryopses defences and attenuate PCD phenomena naturally occurring in the aleurone layer of maturating kernels. AIM OF THE THESIS: Role of Cluster 32 in pathogenesis 1)Production and characterization of Zn2Cys6 knock-out mutant through a plasmid vector We developed an high-throughput gene knock-out system via the TOPO cloning method in order to obtain a Zn2Cys6 knock-out mutant: we have assembled a construct containing the argD gene, coding for Acetyl Ornithine Aminotransferase enzyme, flanked by 3'UTR and 5'UTR, regions homologue to Zn2Cys6. Once obtained, we used the deletion construct to transform AFC-1, a double auxotroph mutant incapable of producing Arginine and Uracil. Simultaneously, to better characterize the metabolic profile related to Cluster 32, we produced overexpression mutants of Zn2Cys6 fused to GFP. Screening was performed basing on fluorescence emission. Such mutants have finally been tested to assay pathogenicity and fitness in different environmental conditions, compared to the wild type with regard to expression profile of some pathogenesis-related genes. After attempting the transformation several times, we were able to isolate three mutants hypothetically carrying our knock-out construct (A1, A2 and B) and two mutants, Arg1 and Arg2, in which the ArgD function had been restored. In order to confirm the positive outcome of the processing and the correct insertion of the construct, we resort to Southern Blotting technique. We performed cDNA amplification of AFLA_096370 together with a subset of genes, of particular relevance, inside the cluster (Salicylate hydroxylase, Zn2Cys6 transcription factor, NPP1 domain protein). This approach should confirm AFLA_096370 deletion as well as its involvement in the regulation of expression of genes inside cluster 32. In fact, the peculiarity of this cluster, is represented by the presence of another transcription factor whose role can synergize or antagonize AFLA_096370, as verified in other SMs clusters as the aflatoxin gene cluster (consider AflJ and AflR for comparison). Regarding information related to the expression profile, data collected from RT-qPCR are, instead, very different: all mutants displayed Zn2Cys6 gene expression and a very similar relative expression values for the other genes analysed. We can hypothesize that the AFLA_096370 deletion cassette has been inserted ectopically (see selective PCR results) and not targeted in a site-specific way the naïve AFLA_096370 locus. In parallel, we attempt to generate an AFLA_096370 over-expressing GFP-fused A. flavus. With the aim of achieving a knowledge that has to be as wide as possible respect to the behavior, to the pathogenicity and to the fitness of the deletion mutants in different environmental conditions, it is necessary to compare these parameters not only to those of the wild type but also to mutants in which the transcription factor of interest is over expressed. Transformation processing has led to the isolation of 15 hypothetical over-expression mutants, which were selected through fluorescence emission. After screening, mutants were singularized on plate, but during this processing, fluorescence extinguished. It is highly probable that, considering how the strain have been transformed and selected, the entire plasmid containing the over-expression-GFP construct was not inserted into fungal genome and was expelled during mycelia duplication. 2) Production of Salycilate hydroxylase knock-out mutant through a plasmid vector Similar to what was done for the transcription factor, a box of destruction was prepared for Salycilate hydroxylase gene. 3) Production of Zn2Cys6 knock-out mutant through Fusion-PCR Overlap extension or fusion PCR is thought to be a simple and easy method to produce fusion DNA fragments without the need for restriction enzyme digestion and DNA ligation. Following Szewczyk et al. (2007), the first PCRs was made to amplify the upstream and downstream region of the pyrithiamine cassette and pyrithiamine itself. Via a first fusion PCR the 5 UTR and the sequence of pyritiamine were combined. Through a second fusion PCR, the previously obtained piece, consisting of two fragments, was attached to the 3 UTR. The risulting construct il about 6000 Kbp. After attempting the transformation several times using Aspergillus flavus NLR3357 protoplasts, we were able to isolate about 20 mutants hypothetically carrying our knock-out construct. To verify the deletion of Zn2Cys6 replaced by pyrithiamine, we made a specific PCR, but the result tell us that the integration is ectopic. 3)Physiological parameters of putative AFLA_096370 mutants (in AFC-1)  Morphological growth WT, AFC-1, knock out and Arg mutants are grown in liquid medium for 10 days in order to acquire information from their morphological behavior (compared to WT and to AFC-1 whose growth is documented). WT and AFC-1 (in PDB and PDB+A+U) have a similar growth even if WT is faster and produces a huge amount of conidia  Aflatoxin production Although Zn2Cys6 is not directly involved in the biosynthesis of aflatoxins, it is plausible that its deletion might have had a more or less sensitive effect even on this pathway and therefore have altered the entire Secondary Metabolism. For this reason a quantitative analysis of aflatoxin, produced by the various mutants and released into the culture filtrate, was conducted. At first detection, by the radiation of aflatoxins suspended in methanol with a UV lamp, it appears that the mutants’ filtrate contains a less amount of aflatoxin instead appreciated in WT, in AFC-1 and in Arg mutant filtrate (where the quantities are similar). B mutant, for example, shows a very few accumulation of aflatoxins in his filtrate whereas A2 filtrate seems even poorer. 4) HPLC analysis of hormones Salycilic acid The aim of this section is the comprehension of crosstalk between A.flavus and Zea mays during the colonization and infection process. In Wt is visible the absence of SA due to the activity of the salicylate hydroxylase which degrade the SA. AFC 1 and mutant B are less virulent or, rather, more slowly in the pathogenesis process. 5) Histological analysis of Zea mays infected with Aspergillus flavus The maize was inoculated with a pin bar wet with the 1 x 106 spores/mL of fungus. After 2, 3 and 4 days the kernels were taken and analysed. At 2 days, Wt has already contaminated the aleurone layer, earlier than the other strains, and its conidia are present within the pericarp while are absent on AFC-1 and mutant B-infected kernels. Also, is remarkable the PCD (programmed cell death) event in all strains. CONCLUSIONS AND FUTURE PERSPECTIVES At the end of the experiments cycles performed to characterize the hypothetical mutants from a molecular point of view and in the light of the results obtained from these, it is plausible to assume that the event of integration of the construct into the genome of A. flavus has occurred. This evidence is supported, in fact, by PCR results and by the phenotypic evidence of recovery of Acetyl Ornithine Aminotransferase activity that allows mutants to survive in minimum media devoid of Arginine. Results obtained by Southern Blotting and RT-qPCR, however, suggest that although the integration has taken place, no locus-specific homologous recombination has occurred in the putative AFLA_096370 deletion mutants. Then, with a high probability, the construct has integrated ectopically within the DNA of A. flavus without removing our gene of interest Zn2Cys6. It is known that A.flavus causes necrosis on infected plants, but in our study we find some interesting structures, one is shown below. These imagine is an appressorium probably indicating a biotrophic phase and then a necrotrophic phase during its infection on Zea mays. The principal target of attack is represented from salicylic acid, implicated in defence response, more than the jasmonic acid (data not shown because not detected). This is demonstrated from the results of qRT-PCR and the HPLC-MS/MS, which support the hypothesis of the importance of salicylate hydroxylase in the degradation of SA to stop the plant defenses. A.flavus is a saprophytic filamentous fungus that is distributed all over the world. It can produced an abundance of secondary metabolites and the most studied is aflatoxins which contaminate crops pre- and post harvest and cause animal and human diseases. This study allows elucidating the infection capacity of A.flavus on corn and could make think strategies control in field to prevent the aflatoxins contamination. This is targeting to limit the risk problem linked to the consummation of corn contaminated by aflatoxins.