We used a transient expression tungsten particle/helium gun bombardment transformation assay to investigate avrRxv function in maize. This assay employed the CRC reporter gene system (a fusion of the R and C1 transcriptional activator genes) that marks cells red due to flavonoid anthocyanin expression. The tissues used were prepared as follows. Mature embryos were derived from germinating scutella and placed face up on an agar medium such that there were 8 scutella in a 2 cm circle (7 peripheral and 1 central), each such octet constituting one bombardment target. Similarly immature embryos were isolated from field-grown ears 9-11 d after fertilization and plated scutellum-side-up on agar medium. Leaves (from stage V5-V6 plants) were obtained from the whorl, unfurled on an agar medium, and covered with a 1.3 cm diameter washer, the exposed center area constituting one target. For more details, see Briggs et al., 1999 PCT WO 99/43923.

When these maize tissues from various genotypic varieties were cotransformed with the avrRxv gene coding region and the CRC reporter system, both driven by the ubiquitin promoter, there frequently occurred suppression of the number of red anthocyanin-labeled cells expressing the CRC reporter gene construct. Table 1 provides quantification of this reduction in CRC expression caused by avrRxv cobombardment. A positive reaction to avrRxv was herein defined as a two-fold or more reduction in the number of red cells. The range in average number of red cells between transformation experiment events could be substantial. Nonetheless, multiple experiments for inbred varieties such as PHN46 clearly indicated that avrRxv caused a marked reduction in CRC expression. The positive reactions to avrRxv varied from 2.0 to 162.9-fold reductions in CRC expression.

Twenty maize varieties were used in these transient transformation studies, 19 inbreds and one hybrid, of which a majority of 12 showed reduction in CRC expression, and 8 did not. The variety 3394 is the F1 progeny of inbred PHN46, which shows the response, and inbred PHP38, which does not. The avrRxv caused a 3.2-fold reduction in CRC expression in 3394, indicating that the response is dominant, or at least semi-dominant. Most of these experiments were done for mature embryos, especially when surveying response in different varieties. However, experiments with PHN46 indicated that this avrRxv response was present in tissues ranging from leaves, immature embryos and mature embryos. In both embryos and leaves, the reduced CRC expression caused by avrRxv in these experiments was correlated with an increase in PR1 protein expression on western blots, a commonly used marker of plant defense system activation.

Maize immature embryo-derived cells were transformed with the ERE::avrRxv construct, which contains an estrogen-regulated promoter (ERE) to drive avrRxv gene expression. This ERE::avrRxv construct contains three tandem plant gene expression units; the estrogen receptor, the estrogen response elements controlling avrRxv, and the selectable marker PAT (phosphinothricin acetyltransferase). This system allows for a tightly controlled, high dynamic range of transgene avrRxv expression, with basal expression permitting recovery of healthy transgenic callus. In ERE::avrRxv transgenic HiII callus, estradiol treatment was demonstrated by northern blots to rapidly (within an hour) induce avrRxv mRNA expression, with a peak by 12-24 hours at 21-fold above baseline. Following estradiol treatment of ERE::avrRxv callus or cell suspensions, there occurred visible and biochemical changes consistent with a precipitant defense response, such as: 1) tissue browning and necrosis; 2) induced expression of methanol-soluble metabolites with an absorbance between 230-350 nm - these likely including phenolics, well-documented plant defense response compounds; 3) induced PR1 and chitinase expression on western blots; and 4) induced cationic peroxidase expression on native IEF gels. The cell death associated with the elevated accumulation of pathogenesis-related proteins and molecules are interpreted as indications that a defense response was chemically-induced in the ERE::avrRxv system. The induction of a cationic peroxidase species by avrRxv was of special interest, because a cationic peroxidase has been shown to be induced in incompatible interactions between rice and Xanthomosas oryzae pv oryzae (Reimers et al., Pl. Phys. 1992 99:1044-1050).

A high density Affymetrix GeneChip® microarray of some 1500 maize gene sequences (est. 1441 unique genes) representing diverse physiological processes, was next used for surveying mRNA expression changes following chemically-induced avrRxv expression in the ERE::avrRxv callus. Protocols for preparing in vitro-transcribed biotinylated cRNA probes from poly-A+ mRNA for Affymetrix GeneChip® gene expression analysis were carried out according to the manufacturer�s recommendations (Affymetrix, Santa Clara, CA). It was observed that estradiol treatment of ERE::avrRxv callus caused a three-fold or more (range 3.5 to 33.2-fold) induction in the expression (mRNA abundance) of nine genes represented on this microarray (Table 2). None of these genes were induced three-fold or more by estradiol treatment of control callus. All of the induced genes are known or suspected plant defense-related genes. In fact, most of these nine have been independently confirmed to be defense inducible in various defense-related experiments, including those with live pathogens, using northerns and other profiling technologies. While one should be aware that promoter systems related to the ERE-estrogen response system could alter gene expression (see Kang et al., Plant J. 1999 20:127-133), these mRNA profiling results, along with the transient bombardment assay above and the previous studies by Whalen et al., appear to support that a maize defense reaction follows induced avrRxv expression. Of the 1500 gene sequences on the microarray, only these nine were induced three-fold or more by these criteria. None of the 1500 genes were repressed three-fold or more. Maize is variously estimated to have between 40,000-80,000 genes, so these 1441 genes represent only about 1.8-3.6% of the total. We can assume by extrapolation that there exist many other co-regulated maize genes not assayed in this study.

By extension from previous related studies involving avirulence gene expression in plants, it would appear that avrRxv elicits an incompatible-like HR response in maize possessing a cognate functional resistance gene (i.e. Zm-Rxv). This putative Zm-Rxv gene appears to be dominant or semi-dominant, like most R genes, and it functions in diverse tissues, namely leaves, mature and immature embryos, and callus/cell suspensions. It is also possible however that the avrRxv defense response is not specifically R-gene or HR-related. Alternatively, at least in maize, AvrRxv could be operating more as a pathogenicity factor that elicits a defense or stress response. Its relationship to YopJ and other pathogenicity or virulence factors, suggests a common and conserved molecular mechanism, one that may have just come to light (see Orth et al. 2000 Science 290:1594-1597).

Of general significance is the observation that an avirulence gene avrRxv, plucked from a specific host-pathogen system, can elicit defense reactions in non-host plants, even those as diverged as monocots and dicots. Together with other transgenic and conventional breeding studies, this appears to affirm an extensive portability of functional resistance and avirulence genes that bodes well for their application in crop improvement. Nonetheless, many avirulence gene types may not be so widely recognized as the group containing avrRxv, which by including YopJ, control defense responses in hosts as far apart as plants and animals.

Table 1. The effect of avrRxv expression on the CRC reporter gene expression (via anthocyanin-producing red cell phenotype), by tissue and genotype.
 

Genotype	Tissuea	Ratiob	DRc
PHN46	ME	90.0	+
PHN46	ME	43.6	+
PHN46	ME	5.0	+
PHN46	ME	28.5	+
PHN46	ME	46.0	+
PHN46	IE	4.1	+
PHN46d	IE	47.0	+
B73	LF	2.0	+
B73	LF	6.9	+
B73	LF	2.2	+
B73	LF	1.6	-e
1047	ME	3.6	+
B73	ME	49.5	+
PHKM0	ME	46.7	+
CN3K7	ME	162.9	+
PH428	ME	4.4	+
PHHB9	ME	4.0	+
953	ME	10.1	+
PHP02	ME	2.0	+
PHP82	ME	17.1	+
PHK03	ME	2.8	+
3394f	ME	3.2	+
PHK76	ME	0.8	-
PHKE1	ME	1.4	-
PHW52	ME	0.9	-
PHW52	ME	1.1	-
PHP38	ME	0.8	-
PHK46	ME	1.1	-
PH647	ME	0.6	-
PHK56	ME	0.9	-
Mo17	ME	0.7	-

^aTissues are: ME, mature embryo; IE, immature embryo; and LF, leaf.
^bRatio of average number red transformed cells for CRC only control divided by the average for CRC+avrRxv cotransformation.
^cWhen the CRC/CRC+avrRxv ratio exceeds two, a defense response (DR) is concluded present (+); when less than two, absent (-).
^dHere only, the avrRxv gene DNA amount 10-fold higher than in the other experiments.
^eRatio falls just below two-fold threshold. here, but other B73 experiments are positive
^f3394 is F1 progeny of PHN46 and PHP38.

Table 2. Affymetrix 1500 GeneChip® Microarray Expression Profiling: mRNAs induced three-fold or more in transgenic ERE::avrRxv cells
 

	Fold Changeb
Gene Name or Descriptiona	Ave	SE
Proteinase inhibitor WIP1 (Bowman-Birk) (=emb|X71396)	33.2	5.9
Chitinase (Class I type) (=EST gi|8273121)	15.1	3.6
Old yellow enzyme homolog (=EST gi|6672185)	11.7	0.2
Proteinase inhibitor (Bowman-Birk) (=EST gi|7031219)	8.7	1.1
Proteinase inhibitor (Subtilisin-chymotrypsin) (=emb|X69972)	7.8	1.1
Major latex protein, root allergen protein (=EST gi|3157104)	4.9	1.7
Antifungal zeamatin-like protein (=gb|U06831)	4.7	0.4
Pathogenesis-related protein (nearly=EST gi|5398728)	4.0	0.3
Metallothionein (=EST gi|5499507)	4.0	0.9

^aGene name or general description derived from blast search of public NR, NT and EST databases. Equal signs (=) indicate essentially exact nucleotide match to the Pioneer Hi-Bred EST gene sequence used on the microarray.

^bFold change in mRNA abundance as measured by relative fluorescent intensity of hybridizing signal. Average and SE were calculated from 3 or 4 replicate comparisons between callus samples ERE::avrRxv estradiol-treated versus control not estradiol-treated.
 
 

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