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In The Super Mario Bros. Super Show! episode "The Mark of Zero", Señor Mayor first appears when Mario, Luigi, Toad, and Princess Toadstool ask the citizens who wanted to help find Zero after she got kidnapped by El Koopitan. After nearly everyone runs away, Señor Mayor explains that the citizens would help find Zero, but El Koopitan strikes fear in the hearts of every one of them.
When El Koopitan enters the village to attack, Señor Mayor and his remaining citizens flee the village and stand right outside to watch the attack take place. When El Koopitan flees after being attacked by Mario, Señor mayor apologizes for fleeing with his citizens explaining that without Zero, everyone is too afraid to fight.
When El Koopitan re-attacks, Mario dressed up as Zero attacks back, which Señor Mayor thinks is the real Zero. With "Zero" back, Señor Mayor and his citizens charge at El Koopitan, Mouser, and his Koopa Troopas demanding liberty and freedom for all fungi.
After defeating El Koopitan, Señor Mayor and his citizens celebrate by tossing their sombreros into the air, not knowing that Zero was still captured.
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