The b-32 ribosome inactivating protein from maize influences fumonisin accumulation in in-vitro bioassays

Fungi of the genus Fusarium are common plant pathogens mainly associated with cereal crops. In particular, Fusarium verticillioides Sacc. is the most common toxigenic fungus in maize worldwide, causing root, stalk, and ear rot. Fusarium spp. can produce a wide range of secondary metabolites, some of which can unfavourably affect human and animal health. Owing to the potential risks of fumonisins F. verticillioides secondary metabolites, new regulations for the allowable mycotoxin limit in food and feed have been put in place by most agencies worldwide. Plants act on the attack of pathogenic fungi through a complex network of active responses such as the production of proteins toxic or inhibitory to pathogens such as RIP (Ribosome-Inactivating-Protein). The RIP present in maize endosperm (termed b-32) has been widely investigated. Similarly to other RIPs, is accumulated in the seed as an inactive pro-RIP precursor, which is converted into an active form by proteolytic processing. To understand the relationships between structure and substrate specificity of the maize b-32 RIP protein, a series of recombinant b-32 sequences, by selective deleting of different domains (RIP b-32, RIP ∆N, RIP ∆C and RIP-∆C (Ala), were prepared. Recombinant sequences were expressed in Escherichia coli to obtain high levels of recombinant proteins, which were subsequently tested for their potential ability to reduce both the colonization of F. verticillioides and fumonisin accumulation

Characterization of the maize b-32 ribosome inactivating protein and its interaction with fungal pathogen development

Plants respond to attack by pathogenic fungi with a complex network of responses, including the production and accumulation of proteins, such as the Ribosome Inactivating Proteins (RIPs) that are toxic or inhibitory to pathogens. In maize endosperm, a cytosolic albumin termed b-32 (RIP1) is synthesized in temporal and quantita¬tive coordination with the deposition of storage proteins. Research has shown that b-32 is able to i) enzymati¬cally inactive ribosomes modifying rRNA inhibiting protein synthesis in vitro, ii) inhibit the growth of Rhizoctonia solani mycelia in an in vitro and in planta assays, iii) reduce Fusarium culmorum head blight in wheat transgenic plants expressing b-32, and iv) diminish Fusarium verticillioides attack symptoms in leaf tissues assays of maize transgenic expressing ectopically b-32 protein. Similarly to other RIPs, maize b-32 is accumulated in the seed as an inactive precursor, which is converted into an active form by proteolytic processing which removes peptide segments from the N (residues 1-16 of pro-RIP) and C (residues 295-301) termini and also from the center (linker domain) of the polypeptide. In this review we will summarize evidence and advances related to the ability of the b-32 protein in contrasting pathogen attacks by considering and describing i) in vivo b-32 antifungal activity and ii) in vitro fungal development inhibition. These data provide information for assessing b-32 in developing plants with a higher capacity to contrast damages induced by pathogens

Spectroscopic Kernel Quality From A Symbiotic Corn Production

The management of the inoculation of a plant's roots, by means of biofertilizers (BF) containing arbuscular mycorrhizal (AM) fungi, is aimed at inducing modifications of the quality of the seeds. It is here shown that a seed-soil treatment can be elicited in the fingerprints of a symbiotic treatment using Near Infra Red (NIR)-SCiO NIR-SCiO spectra collections of single kernels: overall, a sensitivity of 73% and a specificity of 73% have been achieved, thus suggesting that it may be possible to assign the symbiotic origin of corn from just twenty kernels, provided that the dataset is adequately representative of the cultivar and AM. A global correlation study has shown a positive general trend (R2 0.45) of quality vs. quantity, in the sense that an increase in yield corresponded to an increase in the spectral differences between the symbiotic spectra and the control ones, but the inverse was also true, as a result of the parasitic behaviour of the BF treatments. The efficacy of the symbiosis can be back predicted from the NIR spectra; in fact, around 90% of the positive yield outcome results were discriminated from the negative ones. A reduction in the foliar pH (R2 0.37) and an increase in the foliar protein (R2 0.43) were observed as immediate phenotypic signs of a productive symbiosis. The commercial raw composition of the kernels appeared to only be affected slightly by the BF treatments; thus, till now uncharted secondary compounds of the maize kernels are involved, as supported by animal performances

Evaluation of ear rot (Fusarium verticillioides) resistance and fumonisin accumulation in Italian maize inbred lines

Mycotoxin contamination of maize (Zea mays L.) grain is a global threat to the safety of both human food and animal feed. Hence, the development of maize genotypes with reduced mycotoxin accumulation in grain is of major importance. In order to find maize germplasm sources of resistance to Fusarium ear rot, 34 Italian and six public inbred lines were evaluated by means of artificial inoculation in field experiments during 2009 and 2010. Relationships between ear rot and fumonisin concentration in the ears were investigated. Primary ears were challenged with a mixture of two Fusarium verticillioides isolates from Northern Italy, through kernel inoculation, and ear rot severity was assessed.The average number of visibly infected kernels per ear, after inoculation, ranged from 2 to 68 in 2009 and from 0 to 120 in 2010. Fumonisin concentrations in the inoculated ears were greater than in the experimental controls for both years. Variability was found between the inbred lines: fumonisin accumulation ranged from 0.56 to 240.83 mg kg-1 in 2009 and from 1.09 to 190.60 mg kg-1 in 2010. In both years, six inbred lines showed high fumonisin content (≥100 mg kg-1), while the other genotypes were almost equally split into two groups, low (≤10 mg kg-1) and medium (from 11 to 100 mg kg-1) fumonisin content. The number of infected kernels after artificial inoculation correlated with fumonisin concentration both in 2009 (r = 0.94; P≤0.01) and 2010 (r = 0.67; P≤0.01). Additionally, the percentage of internally infected kernels correlated positively with fumonisin concentration (r = 0.37; P≤0.01) and with the number of infected kernels (r = 0.29; P≤0.05). This research has demonstrated that Italian maize germplasm is a valid source of resistance to Fusarium ear rot. Furthermore, there is a strong association of visible Fusarium symptoms with fumonisin concentration, suggesting that selection in maize for reduced visible moulds should reduce the risk of mycotoxin contamination

Multi-Mycotoxin Long-Term Monitoring Survey on North-Italian Maize over an 11-Year Period (2011–2021): The Co-Occurrence of Regulated, Masked and Emerging Mycotoxins and Fungal Metabolites

Maize is considered one of the most susceptible crops to mycotoxin-producing fungi throughout the world, mainly belonging to the Fusarium spp. and Aspergillus spp. Maize is mainly used as animal feeds in Italy, as well as for human consumption, being essential for all the protected designation of origin (DOP) products. Our study investigated the occurrence of regulated mycotoxins in 3769 maize grain samples collected from 88 storage centers by the National Monitoring Network over an 11-year period (2011–2021). Moreover, an in-depth survey over a 4-year period, characterized by extremely different meteorological conditions, was conducted to investigate the co-occurrence of regulated, masked, and emerging mycotoxins. The survey confirmed that Fusarium spp. was the most frequent fungi and fumonisins were the main mycotoxins that were constantly detected in the different years and areas. Moreover, the areas characterized by high fumonisin levels were also the most prone to contamination by emerging mycotoxins produced by the same Fusarium species of the Liseola section. On the other hand, as a result of climatic changes, maize grains have also been affected by the increased frequency of aflatoxin accumulation. Deoxynivalenol, zearalenone, and other emerging mycotoxins produced by the same Fusarium species as the Discolor section occurred more abundantly in some areas in Northern Italy and in years characterized by predisposing meteorological conditions

Co-Occurrence of Moniliformin and Regulated Fusarium Toxins in Maize and Wheat Grown in Italy

The co-occurrence of moniliformin (MON), fumonisins (FBs), and deoxynivalenol (DON) was evaluated in maize, durum, and common wheat grown in different experimental fields located in several Italian regions. MON was quantified using a LC-MS/MS method adding lanthanum ions in the mobile phase. In maize, MON contamination was widespread and considerable; the toxin was detected in almost all the samples (95.1%) and exceeded 500 and 1000 µg kg−1 in 42.0% and in 18.5% of samples, respectively. Significant positive correlation was found between MON and FB contamination levels. When there were not droughty climate conditions, a positive significant correlation was found between growing degree days (GDD) and MON values. In wheat, MON contamination was not widespread like in maize and it was lower in common wheat than in durum wheat. In durum wheat, MON was detected in 45.0% of the samples with only 6 samples (7.5%) exceeding 500 µg kg−1, while in common wheat the toxin was detected above the LOD in 18.7% of samples exceeding 100 µg kg−1 in only two samples (2.5%). No correlation was found with DON contamination. Climate conditions influenced both MON and DON occurrence

Regulation and Processing of Maize Histone Deacetylase Hda1 by Limited Proteolysis

A maize histone deacetylase gene was identified as a homolog of yeast Hda1. The predicted protein corresponds to a previously purified maize deacetylase that is active as a protein monomer with a molecular weight of 48,000 and is expressed in all tissues of germinating embryos. Hda1 is synthesized as an enzymatically inactive protein with an apparent molecular weight of 84,000 that is processed to the active 48-kD form by proteolytic removal of the C-terminal part, presumably via a 65-kD intermediate. The enzymatically inactive 84-kD protein also is part of a 300-kD protein complex of unknown function. The proteolytic cleavage of ZmHda1 is regulated during maize embryo germination in vivo. Expression of the recombinant full-length protein and the 48-kD form confirmed that only the smaller enzyme form is active as a histone deacetylase. In line with this finding, we show that the 48-kD protein is able to repress transcription efficiently in a reporter gene assay, whereas the full-length protein, including the C-terminal part, lacks full repression activity. This report on the processing of Hda1-p84 to enzymatically active Hda1-p48 demonstrates that proteolytic cleavage is a mechanism to regulate the function of Rpd3/Hda1-type histone deacetylases