The resignation of Balwantsinh Rajput (who was also the chief whip), Tejashreeben Patel and Prahlad Patel reduced the Congress's tally to 54 in the 182-seat assembly.
The development came at a time when the Congress has fielded its senior leader Ahmed Patel as its candidate for the Upper House in the election scheduled for August 8.
The Congress nominee can sail through if he musters the support of 47 MLAs and the party is able to guard against further desertions from its legislative arm.
All three MLAs submitted their resignation letters to Assembly Speaker Ramanlal Vora at Gandhinagar.
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"Balwantsinh Rajput, Tejashreeben Patel and Prahlad Patel have given their resignation as sitting MLAs of their respective seats. They now cease to be the members the Legislative Assembly," Vora told PTI.
The trio can't vote in the August 8 Rajya Sabha polls as they are no long members of the House, he said.
Rajput represents Siddhapur seat in Patan district, while Tejashreeben Patel was elected from Viramgam in Ahmedabad district. The third MLA, Prahlad Patel, was elected from Vijapur in Mehasana district.
The latest setback for the Congress comes almost a week after Vaghela left the party and also resigned as the Leader of Opposition. However, he continues to be an MLA.
Ahmed Patel, political secretary of Congress president Sonia Gandhi, yesterday filed his nomination papers from Gujarat for the Rajya Sabha polls.
Of the total 11 Rajya Sabha members from the state, the term of three of them -- Smriti Irani and Dilipbhai Pandya (both BJP) and Ahmed Patel -- is ending on August 18.
Union minister Irani has been re-nominated, while BJP chief Amit Shah will be the other party candidate from Gujarat. Shah is at present an MLA from Gujarat.
After tendering his resignation, Rajput said he was very upset with the Congress due to the allegations being levelled against him.
"I am in public life for 35 years. The people of Siddhapur know how much I have worked for them and for the party. However, it is sad that some persons (in the Congress) have started pointing fingers at me due to my family ties (with Vaghela). That is why I gave my resignation," he said.
"Since the last one-and-a-half years, many of us have been telling the party leaders to give prominent posts to Patels to get their votes in the assembly polls. However, nothing was done in this regard," she said.
Asked about the possibility of her joining the BJP, she said, "We will think about it and take a decision later."
Prahlad Patel said under the Congress "injustice" had been meted out to him and his community.
"I have strengthened the Congress at taluka and district levels. However, injustice was done to me as well as to the Patidar community.
Prahlad Patel indicated he will join the ruling BJP "in the interest of the people".
"Since I belonged to an opposition party, many developmental works were stalled in my constituency. It is an established fact that works are done easily if you are with the ruling party. I am mulling to join the BJP in the interest of my people," he said.