Isabel Murillo

Fungus- and wound-induced accumulation of mRNA containing a class II chitinase of the pathogenesis-related protein 4 (PR-4) family of maize

Pathogenesis-related (PR) proteins are plant proteins that are induced in response to pathogen attack. PR proteins are grouped into independent families based on their sequences and properties. The PR-4 family comprises class I and class II chitinases. We have isolated a full-length cDNA encoding a chitinase from maize which shares a high degree of nucleotide and amino acid sequence homology with the class II chitinases of the PR-4 family of PR proteins. Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by the fungus Fusarium moniliforme, increase the level of ZmPR4 mRNA. In situ mRNA hybridization analysis in sections obtained from fungus-infected germinating embryos revealed that ZmPR4 mRNA accumulation occurs in those cell types that first establish contact with the pathogen. ZmPR4 mRNA accumulation is also stimulated by treatment with silver nitrate whereas the application of the hormones gibberellic acid or acetylsalicylic acid has no effect. Wounding, or treatment with abscisic acid or methyl jasmonate, results in accumulation of ZmPR4 mRNA in maize leaves. Furthermore, the ZmPR4 protein was expressed in Escherichia coli, purified and used to obtain polyclonal antibodies that specifically recognized ZmPR4 in protein extracts from fungus-infected embryos. Accumulation of ZmPR4 mRNA in fungus-infected maize tissues was accompanied by a significant accumulation of the corresponding protein. The possible implications of these findings as part of the general defence response of maize plants against pathogens are discussed.

Cytology of Infection of Maize Seedlings by Fusarium moniliforme and Immunolocalization of the Pathogenesis-Related PRms Protein

ABSTRACT We have investigated the histology of infection of maize seedlings by Fusarium moniliforme in association with a biochemical host defense response, the accumulation of the PRms (pathogenesis-related maize seed) protein. Light microscopy of trypan blue-stained sections and scanning electron microscopy revealed direct penetration by F. moniliforme hyphae through the epidermal cells of the seedling and colonization of the host tissue by inter- and intracellular modes of growth. Pathogen ingress into the infected tissue was associated with the induction of defense-related ultrastructural modifications, as exemplified by the formation of appositions on the outer host cell wall surface, the occlusion of intercellular spaces, and the formation of papillae. Cellular and subcellular immunolocalization studies revealed that PRms accumulated at very high levels in those cells types that represent the first barrier for fungal penetration such as the aleurone layer of germinating seeds and the scutellar epithelial cells of isolated germinating embryos. A highly localized accumulation of PRms within papillae of the inner scutellar parenchyma cells also occurred, suggesting that signaling mechanisms that lead to the accumulation of PRms in papillae of cell types that are distant from the invading pathogen must operate in the infected maize tissues. Our study also revealed the presence of a large number of fungal cells with an abnormal shape that showed PRms-specific labeling. PRms was found to accumulate in clusters over the fungal cell wall. Taken together, the occurrence of PRms in cell types that first establish contact with the pathogen, as well as in papillae, and in association with fungal cell walls suggests that PRms may have a function in the plant defense response.

The development of a rapid PCR assay for detection of Fusarium moniliforme

The fungus Fusarium moniliforme infects a wide range of crops throughout the world. In maize (Zea mays L.) it causes seedling blight and root, stalk, and ear rots. A simple procedure that can be used to detect infection by F. moliliforme from infected plant tissues has been developed. A F. moniliforme genomic library was prepared and used to identify the recombinant clones containing fungal DNA sequences not hybridizing with the DNA of the host plant, maize. Based on the nucleotide sequence information obtained from the F. moniliforme pUCF2 genomic clone, specific oligonucleotides were designed and used as primers for in vitro DNA amplification by the polymerase chain reaction. An amplification product was obtained with F. moniliforme DNA preparations whereas no amplified DNA was detected with DNAs from other fungal pathogens, including various Fusarium species, or from the host plant. This PCR analysis was successfully employed to identify F. moniliforme directly from the mycelia that develop from naturally infected maize seeds, with no need to obtain pure fungal cultures for reliable diagnosis. The protocol can be used for the diagnosis of infected plants and soils in epidemiological studies of Fusarium diseases, for seed health testing, and for evaluation of susceptibility to colonization in commercial maize hybrids.

Use of oligonucleotide-alkaline phosphatase conjugates as non-radioactive probes for rapid analysis of a proteinase inhibitor gene fromZea mays

Conjugates of oligonucleotides and alkaline phosphatase have been prepared and used as nonradioactive hybridization probes for the study ofPis3 (=MPI) a gene encoding a proteinase inhibitor fromZea mays. Attachment of the alkaline phosphatase was carried out either at the 5′ or 3′ end of two 25-bp oligonucleotides. Sensitivity of each alkaline phosphatase-oligonucleotide probe was assessed using a chemiluminescent substrate for detection of alkaline phosphatase activity. This sensitive method allows the rapid analysis of genomic clones isolated from aZea mays library and the subsequent characterization of the completePis3 gene without the need for construction of restriction maps for the cloned DNA fragments. This general strategy may be valuable for the identification of any gene for which a limited sequence is known and for location of specific DNA sequences that represent a small region within a larger DNA fragment.