I created an eo.po in /usr/src/build/rt-3.5.5 and re-ran make regenerate-message-catalogs.
And hurrah, eo.po was populated. Can I simply edit this file and place it in /opt/rt3/lib/RT/I18N or is that to simple?
It is that simple. However, you should give a try to kbabel which is way easier to edit .po files. It also has useful checks about the .po, such punctuation, right number of placeholer in translated string compared to the original one, and so on.
Do not forget to "msgfmt eo.po" to check syntax if you edit it by hand.