Maize
Genetics Cooperation Newsletter 80. 2006.
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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note:
Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with
consent of the authors.
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