Maize Genetics Cooperation Newsletter 80. 2006.

 

The maize ribosome-inactivating protein b-32:  role in the defence against fungal pathogens

--Balconi, C; Lupotto, E; Triulzi, T; Lanzanova, C; Gualdi, L; Conti, E; Motto, M

 

       In maize endosperm, a cytosolic albumin with a molecular weight of 32 kDa, termed b-32, is synthesized in temporal and quantitative coordination with the deposition of storage proteins (Soave et al., Cell 27:403-10, 1981).  Both cDNA and genomic clones encoding b-32 have been isolated.  It was shown that the b-32 genes form a small gene family (Hartings et al., Genet. Res. Camb. 65:11-19, 1995).  The b32 gene, as well as the 22 kDa storage protein zeins, are under the control of the seed-specific transcriptional activator opaque2 (o2).  In opaque2 mutants the b-32 protein is expressed at very low levels (Lohmer et al., EMBO J. 10:617-24, 1991).  Although the role of b-32 in maize endosperm remains unclear, this protein has homology with several previously characterized ribosome-inactivating proteins (RIPs).  It was found that b-32 is a functional RIP by the criteria of inhibition of in vitro translation in a cell-free rabbit reticulocyte system and specific N-glycosidase activity on 28S rRNA (Maddaloni et al., J. Genet. Breed. 45:377-80, 1991; Bass et al., Plant Cell 4:225-34, 1992).  Additional evidence indicated that transgenic tobacco plants expressing b-32 showed an increased tolerance against infection by the soil-borne fungal pathogen Rhizoctonia solani (Maddaloni at al., Transg. Res. 6:393-402, 1997).

       Research is in progress in our laboratories to verify if maize plants expressing b-32 in various organs and tissues have an increased defence against fungal pathogens in comparison with plants expressing b-32 only in the kernel.  For these purposes transgenic plants were obtained through genetic transformation using the vector pSC1b32 containing the b-32 coding sequence of clone b32.66 under the constitutive promoter 35SCaMV and the cassette ubi1-bar for Basta herbicide resistance as a selectable marker.  These plants (T0) derived from callus regeneration after in vitro selection were tested for resistance to the herbicide Basta.  T0 (Tt) plants were pollinated by B73 (tt) plants to obtain T1 plants.  T1 seedlings were sprayed with Basta at the 3-leaf stage, and resistant plants were grown to maturity in a controlled environment.  T1 plants were self-pollinated and were fully fertile and set seeds.  In addition, biochemical analyses at the stage of flowering showed expression of the engineered protein in b-32 transgenic plants in tissues likely to be target sites for fungal invasion: silks, rachis, brace roots and husks, in addition to leaf tissues (Lanzanova et al., MNL 77:7-8, 2003).

       A set of progenies (SM1, SM3, SM4, SM16, SM19, SM20) PCR b32+ and western+, and the progeny SM8 PCR b32+ and western negative (i.e., the 35S-b32 cassette is integrated but b-32 is not expressed) have been used in our studies.  A detailed analysis of b-32 expression in leaves and pathogenicity tests were performed on the progenies indicated above.  Twenty seeds from each SM progeny were sown in a controlled environment and the tt plants eliminated by Basta spraying.  Tt and/or TT plants were raised to maturity in a containment greenhouse.  At flowering, two individuals for each progeny were analysed for the expression of b-32 in leaf tissues and for response to Fusarium verticillioides attack.  The expression of b-32 was analysed in immuno-blot assays.  As expected, in SM8 progeny b-32 expression was not detected; therefore, this progeny represented our negative control for pathogenicity tests.  On the other hand, all the other progenies tested were b32-western positive.  Comparison of b-32 expression among various individuals was performed after immuno-blot imagine scanner acquisition, using IMAGINE MASTER 1D Elite Version 3.01 (NonLinear Dynamyc Ltd) software.  A differential b-32 expression in the various progenies was recorded; SM1 progeny showed the highest b-32 expression (SM1.1, the individual with the most abundant b-32 content in leaves, was chosen as reference for b-32 relative abundance in leaves); SM3.1 showed 89% b-32 content in comparison to SM1.1; SM20 and SM4 progenies had around 70% and 50% b-32 content, respectively, in comparison to SM1.1.  All individuals belonging to SM16 and SM19 progenies possessed a b-32 content lower than 25% with respect to SM1.1.

       This analysis allowed the identification of progenies with high (SM1, SM3, SM20), intermediate (SM4), and low b-32 (SM16, SM19) expression in leaves at the flowering stage; this is a useful range of expression for pathogenicity experiments to evaluate a differential response to Fusarium attack in leaf tissue colonization bioassays.  For this purpose, at flowering stage, leaves of SM8 progeny not expressing b-32 protein were collected to determine the optimal spore concentration to generate a detectable Fusarium attack.  Fusarium verticillioides (MRC826 strain, supplied by PRI-Wageningen) was grown on Potato Dextrose Agar (PDA) plates at 26�C until the mycelium covered the surface of the plate and used for fresh spore inoculum production.  Leaves were surface sterilized and segments (1 cm squares) were dissected.  Four leaf squares were plated on PDA and inoculated with a 5�l spore suspension at four different concentrations: (104, 105, 106 and 107/ml).  At least three replicates for each experimental condition were considered.  Infection progression was detected at different days after inoculation; mycelial growth was monitored daily, measuring fungal colony diameter around inoculated leaf squares.  Control leaf squares were non-infected or treated with sterile water.  This preliminary experiment supported the choice of a 105 spore/ml concentration, and of 3-5 days after inoculation as the detection time, for a reliable evaluation of the responses.  Using this spore concentration, fungal growth on the leaves was gradually visible up to four days following the inoculation.  Later on, the diameter was too large to be correctly measured because of colony confluence around the leaf squares plated together.  In addition, when leaves were inoculated with suspension containing 105 spores/ml, mycelia were evident on the cut edges of leaves within 3 days, and later also on leaf surfaces.  This last observation was not evident inoculating leaves with a spore concentration of 104/ml.  Both controls, non-inoculated and sterile water-inoculated leaves, did not show any mycelial growth.  Therefore, the protocol adopted for leaf tissue surface sterilization appeared to eliminate all external contaminations.

       The bioassay parameters described above (5�l suspension containing 105spores/ml, 3-5 days following inoculation as detection time) were adopted for pathogenicity experiments including the negative control SM8, in addition to individuals of progenies previously tested for b-32 expression in leaves.  Results indicated that the negative control SM8, when evaluated 4-5 days after inoculation, was more susceptible to Fusarium attack, in comparison to all the other progenies tested.  At this stage, fungal colony diameter measured around the inoculated leaves of SM8 was significantly larger than that observed in progenies expressing b-32.

       A good correlation between b-32 content in the leaves and level of resistance to Fusarium attack was observed.  Individuals belonging to SM16 and SM19 progenies, with a b-32 content in leaves lower than that detected in the other progenies, appeared to possess a level of resistance to Fusarium significantly lower, showing high mycelial growth around inoculated leaves (colony diameter �15 mm, 5 days after inoculation).  In addition, for these progenies (SM16 and SM19) and for the negative control SM8, a clear extension of mycelial growth on leaf surfaces was observed 3-4 days after inoculation.  In the case of progenies with high (SM1, SM3, SM20) or intermediate (SM4) b-32 content in the leaves, in addition to a reduced mycelial growth on the cut edges of the leaves (colony diameter �10mm), a reduced growth on leaf surfaces was observed, even more than 5 days following inoculation.  Experiments are in progress to extend pathogenicity tests to other plant tissues and to evaluate the specificity of b-32�s role in the defence against other fungal pathogens (i.e., Aspergillus, Penicillium).

       *This work has been developed within the framework of the EU-funded project SAFEMAIZE (ICA4-CT2000-30033) in FP5.

 

 

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